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ScanArray Express Microarray Analysis System User Manual

1. Palette Source Black threshold ee o SPES Ful color threshokt 33 Median Auto Adjust Mouse is at 16823m 21602um Pixel intensity ratio 1 91 2 3 1 Improving the Image Display You can use the controls in the lower right corner to adjust the image display and improve the visibility The adjustments do not affect your data only the image on the screen Note ScanArray Express auto adjusts images during scanning or opening The image of the Geometric Test Slide that you have just scanned using parameters in the previous section does not need adjustments The image is already of good quality However if you want to see at this point how the adjustments work you can find more information in Adjusting the Display in Chapter 3 2 3 2 Changing the Palette for the Selected Image You can select a different palette or change the contrast The palette used in the composite tab cannot be changed it is always a blending of the palettes of the control and source experiment images or the rainbow palette If you open a palette when the Composite tab is selected the Palette window that opens allows you only to adjust the contrast User Manual pbtsupport perkinelmer com 2 5 Chapter 2 2 4 Getting Started Scan and Quantitate the Click Palette The Set Palette Options window opens Set Palette Options Name Palette Red Orange Yellow Blue Purple Gray Rainbow Use the pale
2. 5 Tools Scan resolution Sum 10 um 20 um C 30 um 50 um Help and Directions Focus position should generally be 0 pm Scan resolution should be about 1 10th of the spot diameter Scanspeed should be Full unless you specify a resolution of 5 um or unless you particularly desire low signal to noise ratios at high PMT gains Finish Cancel Figure 4 4 Scan Protocol Basic Information Window Click Next to move to the next window and Back to move to the previous window Click Finish to save all of your changes and close the wizard Click Cancel to close the wizard without saving any of your changes 4 4 1 Basic Information In the Scan Protocol Basic Information window provide a name and description for the protocol and choose the scanning resolution and speed 4 6 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 To name a scan protocol 1 Fill in the information as listed in the following table Item Description Name Description Focus Position Scan Resolution Scan Speed Enter a name for the scan protocol so you can recall it easily You must enter a name in order to proceed Enter a description for the protocol You do not need to adjust this for each scan Leave the default value at 0 for conventional flat slide substrates To determine the focus position for unconventional slides use the Scan Protocol Tools
3. M Use Cy3 Scan Area Co ordinates Start position X mm 1 04 Area width mm 7 52 Start position Y mm 1292 Area height mm 8 23 Set Scan Area to Full Microarray Show Zoom Window J Automatically save image files locally To change the scan area drag J Automatically save images in Arraylnformatics the rectangle edges with the mouse or use the the box labelled Area Co ordinates at Cancel right a Figure 3 1 Scan Window Run Easy Scan 3 Use the settings from the last session if you are using the same type of microarray or see Section 3 2 1 to change the settings 4 Click Start You may or may not see a message asking if you want to skip warming up the lasers Skipping the warmup does not cause any damage but the sensitivity of the resulting scans will vary slightly If you are concerned with precise repeatability or uniformity of scanning wait for the lasers to warm up 3 2 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express User Manual Using Easy Scan and Easy Quant Changing the Settings Chapter 3 To change the Easy Scan settings refer to the table below for information To make it easier to select a scan area using the mouse you may want to obtain an image first by scanning the full slide at 50 microns Item Description Scan Type Scan resolution Autoloader slot from which to load Fluorophores Use PMT Gain Scan Area Area Co or
4. access to your instrument your phone number fax number and e mail address For Additional Information Additional information about the system can be found in the following ScanArray Express Installation Guide e Documentation provided with the workstation and its operating software Documentation provided by manufacturer of the fluorophores that are used e The ArrayInformatics Microarray Laboratory Integration Guide for ArrayInformatics users only Preface Preface Getting Help for Windows 2000 XP The Windows 2000 XP online help provides information on learning how to use the Windows operating system To open Windows 2000 XP help 1 Click the Start button on the task bar 2 On the Start menu click Help Navigate to a topic using the tabs User Manual PBTsupport perkinelmer com Pagex Preface Preface Introduction Ciapies 1 Chapter Summary Overview 1 1 The ScanArray Express Main Window 1 4 Workflow for Scanning and Quantitation 1 5 The Different Ways to Scan 1 6 The Different Ways to Quantitate 1 7 Preparing to Scan 1 8 1 1 Overview Welcome to the ScanArray Express from PerkinElmer Life Sciences The ScanArray Express is an extremely sensitive microarray laser scanner with easy to use software for microarray scanning and quantitation 1 1 1 The ScanArray Express System The ScanArray Express system includes the scanning instrument with one to four lasers and a Windows 2000 or Windows XP wor
5. Select a resolution that is appropriate for your microarrays 5 um for spots that are less than 90 um diameter 10 um for spots that are 100 to 200 um diameter 20 ums for spots that are greater than 200 um diameter Higher settings result in a quicker image with reduced detail The default is 10 microns Select a scan speed usually Full the default setting You may want to use Half speed for better signal to noise ratio on dim arrays that require PMT settings greater than 75 2 Click Next to display the Scan Protocol Scan Area window 4 4 2 Selecting the Scan Area In the Scan Protocol Scan Area window select the area of the slide to be scanned To set the scan area to include only that part of the slide with the image obtain an image to help you see the area There are two ways you can get an image e The first is to run an Easy Scan on your slide before you start to configure the scan protocol The second if you ve already started configuring the protocol is to run a Quick Scan from the Tools section of this protocol See Running the Quick Scan on page 4 15 To select the Scan Area 1 Open the Scan Protocol Scan Area window shown in Figure 4 5 User Manual pbtsupport perkinelmer com 4 7 Chapter 4 Scanning with Protocols Scan Protocol Scan Area J asic Information Help amp Directions The area to be scanned is shown as a darted an Area rectangle J 3 Fluorophores To Start a Ne
6. Change after selecting a protocol to modify an existing protocol Duplicate makes a duplicate of the highlighted protocol to help you create a new protocol from one that is already close to what you want Or click e Delete after selecting a protocol to delete a protocol you cannot delete a protocol that is part of a batch set or protocol group e View Usage after selecting a protocol to display a list of batch sets and protocol groups which include the selected protocol 4 4 The Scan Protocol Wizard The Scan Protocol wizard opens with the Basic Information window displayed as shown in Figure 4 4 From this window using the wizard you can display and set all of the scanning parameters User Manual pbtsupport perkinelmer com 4 5 Chapter 4 Scanning with Protocols The only required steps in the wizard are 1 Basic Information and 3 Fluorophores At least one fluorophore must be added to a new protocol for it to be created and saved Tip You may want to run an Easy Scan to acquire an image before configuring your A scan protocol to help in setting up the protocol You can move through the windows by clicking the numbers on the left or by using the buttons on the bottom of the window Scan Protocol Basic Information Click the number to move f from one window display to another gt 2 ScanArea 1 Basic Information Name i Description 3 Fluorophores Focus position pm o
7. Fulwidh Zoom to Line CR cancel Figure 4 17 Focus Line Definition Window 3 Click OK to close and return to the Automatic Focus Calibration window 4 Click Start A progress bar displays while the focus is being calibrated You can cancel at any time by clicking Stop 5 Upon completion of the calibration review the results and click Accept Changes to accept the results Note If you have used the Scan Protocol Tools and accepted changes made by the tools be sure to save your Scan Protocol to save the changes User Manual pbtsupport perkinelmer com 4 23 Chapter 4 Scanning with Protocols 4 6 Creating an Image Autosave Protocol Image Autosave Protocols specify the folder file naming convention and file type to use when automatically saving image files during a scan To create an Image Autosave Protocol 1 On the Main Window click Configure in the Configure amp File group In the Configure menu that displays click Image Autosave Protocols The List of Image Autosave Protocols window opens List of Image Autosave Protocols Description Pattern Default lt Fluor lt Date gt lt Time gt C Program Files PerkinElr Change Duplicate Delete View Usage Sort By Name Description Path mage autosave protocols beginning with cannot be executed with current hardware Figure 4 18 List of Image Autosave Protocols Window 2 Sort the list optional by Name Description or Path
8. n 34s siete es Soin Soe ee dw bet ee Rene a ORS 7 12 Backing up the Database 24 27o ee ah ene ee ee kaye wate e eae 7 13 Restoring the Databases 43 4ncdcuc peodined ch ei deud paweede wp bads 7 13 S rvice Featuics Vs cachid wies Gea dees weed ade aaa Ai aw ene Bae 7 14 Descriptions essea ra he pha iy Dewees ee ak ed aaa a ee 7 14 Setting the Instrument Network Options 00 0002 eee eee 7 16 Chapter 8 Diagnostics and Troubleshooting OVETVIEW aa sone ia o E See gre wndeee hed Ge nied Bag wines peg dali deg gaia 8 1 Diagnostic OOS eosar tee cee hued ea RG ie yea we ane Rao he a 8 1 Start up DigenOsics 6 004 aye ote er ek 2G te el Ma rad aan iG 8 1 Instrument Statistics Accumulation 0 ce eee eee eee eee 8 3 Troubleshooting 2 id 0c trv Porat a ulate cate win E eu le Aare ey ta se WD Ore aes oa ae 8 3 Hard Wares aia oo aeg tk oa yas TEE GIA Cees SSy eee wtesa 8 3 Ready LED Flashes Yellow e336 e853 d VEGA PARR SYS RRE SSPE RRS OS 8 3 External Laser Will Not Turn On 0 000 tenes 8 3 Microarray is Jammed ccs seta ais ece ua nl den nel a ihe dee Dey doe Satins ca 8 3 Poor Image Uniformity oii Pork Oh oeren ea ee ook Gi SOE ON a Ree 8 4 BOL Wale roc sealer eRe eee eats Matha a BAGS Hae S SE Le eee s TAATA 8 4 Unable to Open Stored TIFF flesh S23 woke 4 cagdon ee tala nde eae 8 4 Recording the ScanArray Express System Activity 0 0 0 eee eee ee 8 4 Viewing the System Log 44044 seceded ada eoa a
9. on page 1 6 e Easy Scan for ways to scan and save your images e Scan Protocol See Chapter 3 to get started right away with e Protocol Group Easy Scan e Batch Set gt See Chapter 4 to create a Scan Protocol or Save your images manually or Chapter 6 for a Protocol Group or Batch Set automatically Quantitate See Different Ways to Quantitate on page Click Quantitate and select from 1 7 for ways to quantitate and save your data e Easy Quant e Quantitation Protocol gt See Chapter 3 to get started right away with Easy Quant See Chapter 5 to create a Quantitation Protocol Quantitate Automatically Save your images or quantitate See Chapter 4 to run a Scan Protocol with a automatically after scanning and then save checkbox for automatic quantitation the images and quantitation results NOTE To practice scanning with the Geometric Test Slide included with your system see Chapter 2 Getting Started Scan and Quantitate the Geometric Test Slide Figure 1 3 A Workflow Roadmap User Manual pbtsupport perkinelmer com 1 5 Chapter 1 Introduction 1 4 The Different Ways to Scan Before starting review the ways you can scan your microarrays You can automatically save the images to a local network file system or ArrayInformatics database 1 4 1 Easy Scan The Easy Scan settings are persistent that is they remain from the last scanning session so it s easier to scan the same type of microarra
10. 1 Click the ArrayInformatics tab Application Settings Connection Scanning Quantitation Arraylnformatics Settings Database Lspg dey Image path SLspa dev lmageStore Browse Image path to use when Arraylnformatics is unavailable IE ImageT empStore Browse 2 Refer to the following table and specify your settings Item Description Database This field displays the database that is currently active for this ScanArray Express system The database cannot be changed from this window but must be changed using Array Informatics Database Tools Image Path Browse the network to locate and select the directory that has been set up for saving the scan images Images that are saved to Arraylnformatics are saved to this location Image Path to use This is a local directory where scanned images are saved if the when Arraylnformatics is temporarily unavailable or some other error Arraylnformatics is prevents saving in ArrayInformatics Images that are saved to this unavailable directory can later be sent to the Arraylnformatics database pbtsupport perkinelmer com 7 5 Chapter 7 System Settings 7 2 5 Other Settings Settings on the Other tab include those that affect what is seen on the user interface To set or change these settings 1 Click the Other tab Application Settings Connection Scanning Quantitation Arr Informatics Image Tab Settings Action performed by clicking mouse on image
11. 3 Click one of the following in the List of Image Autosave Protocols window e Add to create a new protocol e Change after selecting a protocol to modify an existing protocol Duplicate makes a duplicate of the highlighted protocol to help you create a new protocol from one that is already close to what you want Delete after selecting a protocol to delete it e View Usage after selecting a protocol to display a list of protocol groups and batch sets which include the selected protocol 4 24 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 4 Click Add The mage Autosave Protocol dialog box displays In this window you can check each item that you want to include in the filename To create a unique filename you must include the fluorophore the date and the time You can also check Custom Text and add a version number e g v1 v2 etc Image Autosave Protocol Description Pattern for File Names Information to include in the file names IV Fluorophore J Time hours minutes J Barcode J Custom text IV Date month day year File name pattern lt Fluor gt lt Date gt File types Tl BMP bitmap J JPEG Joint Photographic Experts Group J RAW proprietary binary format IV TIFF Tagged Image File Path to output files C Program Files PerkinElmer Scan4rray Express lm Browse Car Figure 4 19 Image Autosave Pro
12. 7 3 Configuring Fluorophores Fluorophores specify the laser wavelength and emission filter used to scan a microarray The ScanArray Express ships with a number of pre defined system fluorophores These cannot be deleted and only the display palette field assigned to the fluorophore may be changed User defined fluorophores may be added duplicated changed and deleted as specified below A fluorophore currently specified in a protocol may not be deleted 7 3 1 Defining a Fluorophore To add a new fluorophore to ScanArray Express 1 On the Main Window click Configure in the Configure amp File group then click Fluorophores The List of Fluorophores window displays List of Fluorophores Pe Excitation Emission Composite Image Description Peak nm Peak nm Palette Palette Standard Alexa 438 Alexa 488 Alexa 532 Alexa 532 Alexa 546 Alexa 546 Alexa 555 Alexa 555 Alexa 568 Alexa 568 Alexa 594 Alexa 594 Alexa 647 Alexa 647 Alexa 660 Alexa 660 Allophycocyanin APC Allophycocyanin APC BODIPY 530 550 BODIPY 530 550 BODIPY 558 563 BODIPY 558 563 BODIPY 564 570 BODIPY 564 570 BODIPY 630 650 BODIPY 630 650 BODIPY TMR BODIPY TMR Calcein Calcein Calcium Crimson Calcium Crimson Calcium Green 1 Calcium Green 1 Calcium Orange Calcium Orange cy 3s cy 3s cys cy ss Cy2 Cy2 Cy3 Cy3 Delete View Usage Sort By Name Description Excitation Peak Emission Peak q lt lt lt lt lt lt lt lt lt
13. Centers point Centers point and zooms in Centers point and zooms out Program Layout nstrument controller layout Quantitation only layout Item Visibility J Hide items protocols protocol groups batch sets and fluorophores that are incompatible with current hardware IV Hide diagnostic log messages 2 Refer to the following table and specify your settings Item Description Image Tab Settings Select a method to set the behavior of the mouse click on all image tabs Action performed by Centers point mouse click on image Centers point and zooms in Centers point and zooms out Program Layout Select whether the ScanArray Express system is to show both scanning and quantitation controls or only quantitation controls Instrument Controller layout Quantitation Only layout the user see only Quantitation buttons and windows The scan button does not display Item Visibility Check one or both boxes Hide items protocols protocol groups batch sets and fluorophores that are incompatible with current hardware If this box is not checked the incompatible items display marked with an asterisk Hide diagnostic log Keeps the diagnostic instrument messages from displaying in the Log messages tab on the Main Window 7 6 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Settings Chapter 7 3 Click OK to save changes and close the Application Settings dialog box
14. AN Note For instruments with HT capability specify the slot number of the microarray to be scanned before selecting a tool to run The default slot value is 1 2 After using the Tools to refine settings click Finish to save the protocol 4 5 Using Scan Protocol Tools To use the tools run a Quick Scan first only experienced users should run the other tools 4 5 1 Running the Quick Scan User Manual The Quick Scan allows you to check the setup of the protocol Any changes made during a Quick Scan do not persist unless you save them to the protocol The Quick Scan performs a scan of a selected area using selected fluorophores allowing you to adjust the laser power and PMT gain while the scan is being performed pbtsupport perkinelmer com 4 15 Chapter 4 Scanning with Protocols To perform a Quick Scan 1 Click Run in the Quick Scan area of the Scan Protocol Tools window The Quick Scan dialog box displays Quick Scan Area to Scan Scan resolution jm C5 10 C 20 30 50 Focus position um l PMT Laser Fluorophores Gain Power Cy3 Cy5 Select a Fluorophore l Select a Fluorophore Select a Fluorophore l Help amp Directions The area to scan is shown as a darted rectangle For instructions on how to select an area click Show Zoom Window Show Zoom Window Cancel Figure 4 12 Quick Scan Window 2 Change settings only if needed Refer to the following t
15. Chapter 7 System Settings Pre printed barcode labels are commercially available or you can print your own that conform to the guidelines in Table 7 1 PerkinElmer has qualified the following blank labels Manufacturer Part Number Size inches Size mm Substrate Intermec E17512 0 920 x 0 550 23 4 x 14mm The label containing the barcode should fit on the first 15 mm of the slide so that it does not tilt the slide in the slide holder and the label must be centered on the slide PerkinElmer has qualified a printer and labels that will withstand hybridization washing and other processes The recommended printer is an Intermec 3240 with a 137071206 Super Premium ribbon The recommended label also from Intermec is L3203011 2 mil glass poly with dimensions of 0 920 x 0 550 and a corner radius of 0 04 to 0 06 Information about the printer and the labels can be obtained from Intermec Internet Address http www intermec com e mail info intermec com You must define the barcode parameters which can include the Image Autosave Protocol and Scan Protocol as described in the next section Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Settings Chapter 7 7 4 2 Defining Barcode Parameters To define barcode parameters 1 On the Main Window click Configure in the Configure amp File group and then click Barcodes to display the Barcodes window Barcodes IV Barcodes contain image
16. Each row in the spreadsheet is the data for one spot If you use a GAL file for your template the gene name and ID are imported for each spot Run Composite Cy5 Control Cy3 Source Spreadsheet Scatter Plot Distribution Piot Log Diagnostics Help Scan General CyB Control Raw Spot CyB Cont it Subanay Spot Spot Spot Spot gt gt Quantitate ity Lacanen fied mn cee DNa eons iscaten re Meden Mem Medan en eed ean ee StDev Bkgnd Bkond fecliow coiro fx umy pm z pot Spot Bkgnd Bkgnd z 7 18 28 PRESAS A 1 11 1 1 G 0346 39706 640 5 100 0 00 Configure amp File f2 11 2 1 AKTI Hs 71816 1453 13837 Good 48027 4026 47365 39464 662 1292 14458 56 480853 909 SDS me fi Ww 3 1 AKT2 He 200816 1659 13837 Good 42628 40464 42016 39852 612 608 1445261 12974 1000 1000 E 4 XI 41 AREG Hs 1257 1859 13837 Good 4780 40510 46573 39903 607 615 1496348 16831 1000 100 Configure 5 Ww 5 1 ASPH Hs 121576 2069 13837 Good 46629 40045 46083 39499 546 583 1436290 15551 1000 100 aome 5 4 6 1 BAGI Hs41714 2269 13837 Good 48168 40223 47598 393 592 608 1441559 15582 1000 100C 7 li 7 1 BCGFI Hs 99879 2469 13837 Good 47io7 39410 46528 aeeai 579 65 1388111 14225 1000 1006 f 8 11 8 1 BCL2 Hs 79241 2679 13837 Good 48668 45410 48071 44813 597 593 10213 38 148 09 100 0 100 0 Spot Viewer 3 WwW 3 1 BDNF Hs 56023 2879 13837 Good 50843 47266 50227 46650 616 626 1044249
17. Spot Pile Encel Spreadsheet None Figure 3 6 Quantitation Results Tab You can scroll down t o see all of the listed spots or scroll across to see all columns of data click View as Excel Spreadsheet to view and save as a Microsoft Excel spreadsheet Several spot intensity values and ratios between multiple fluorophores are listed in the spreadsheet Spot intensity values are automatically normalized to correct the intensity of each spot for variations in the overall intensity of the image with respect to a control image Values include Value Description Median Mean User Manual pbtsupport perkinelmer com This column normalizes the ratio of the median value of all spots in each fluorophore to the ratio of the median value of the spots in the control fluorophore This column normalizes the ratio of the mean value of all spots in each fluorophore to the ratio of the mean value of the spots in the control fluorophore 3 15 Chapter 3 Using Easy Scan and Easy Quant Value Description Total Each spot s data is normalized to the data of all spots Background Reports the intensity of each spot in the array minus the background for Subtracted that spot 3 4 1 1 Filtering the Spreadsheet Data You can use the buttons at the bottom of the Spreadsheet tab to filter the data as described in this section Show Hide Show Hide Set Quality Set View Spots by Status Columns Cri
18. is the physical object an array is an ordered collection of spots on the microarray A network interface card NIC is a computer workstation circuit board or card that is installed in a computer so that it can be connected to a network Workstations on local area networks LANs typically contain a network interface card specifically designed for the LAN transmission technology such as Ethernet A range of colors assigned to image pixels based on the intensity value for the pixel The loss of fluorescent signal due to intense laser power This can be minimized by lowering laser power and by subjecting the sample to the laser power for less time The lower the resolution the less time the microarray is subjected to the lasers At a 10 micron scan with good quality dyes and 95 laser power typical signal loss is less than 2 to 3 per scan Images are made up of a 2 dimensional array of numbers called picture elements or pixels The resolution of an image is determined by the size of pixels For example 5um pixels is higher resolution than 10um Periodic Maintenance and Inspection Planned maintenance calibration and inspection to correct for expected wear in the system Photo Multiplier Tube A light sensitive vacuum tube that controls current based on the amount of light being received by the tube A PMT has high gain allowing it to have a high currentto Light ratio The PMT absorbs low level fluorescence light and converts it t
19. see Appendix A The template field fills with the name of the selected GAL file See To define a template using a GAL file on page 3 10 Click this button to manually define the array pattern The Template Specification window opens where you enter the values See To define a template from specifications on page 3 11 Click this button to import template information from the Arraylnformatics database if your ScanArray Express system is integrated with ArrayInformatics Refer to the Microarray Laboratory Integration Manual that ships with your ArrayInformatics software If your system is not integrated with Arraylnformatics this button appears dimmed and is unavailable to select Select Adaptive Circle This is the default method For more information on quantitation methods see Choosing a Quantitation Method in Chapter 5 Select LOWESS This is the default method For more information on normalization methods see Chapter 5 Click this button to open the Adjust Template window to adjust the template and register the images if there are two images When you start the quantitation ScanArray Express finds the spots using the template and then starts quantitation For fastest spot finding adjust the template within a row or column or two of the microarray spots See Adjusting the Template and Registering Images on page 3 12 User Manual pbtsupport perkinelmer com 3 9 Chapter 3 Using Easy Scan and Easy Qua
20. which is adequate for pre scanning and investigative scanning After five minutes they become stable enough for all but the most sensitive applications ScanArray Express indicates the lasers are still warming up for the full 15 minutes 1 6 1 Turning on the ScanArray Express and Warming the Lasers To turn on the ScanArray Express 1 Turn on the ScanArray instrument Verify that the Power and Ready indicator lights are green os Check that the two Hold the microarray indicators are green face up by the thumb space or by i k the edges 2 Log in to Windows if required using your assigned user name and password 3 On the Windows workstation double click the ScanArray Express icon Scandrray Express The User Interface opens to the ScanArray Main Window 4 Check the status of the lasers The laser buttons on the right side of the Main Window indicate the status of the lasers blue indicates the lasers are turned off yellow indicates the lasers are warming up green indicates the laser are ready To turn on a laser click the status button for that laser 1 8 Technical Support 800 551 2121 or 617 350 9263 ScanArray TMEX press Introduction 5 Chapter 1 Begin laser warm up by clicking the laser status button 1 6 1 1 Loading the Microarray or Cassette To load a microarray 1 2 Hold the microarray by the thumbspace or by the edges Holding the microarray face up and with th
21. 18850 1000 100C iad 10 1 101 BMP2 Hs 73853 3069 13837 Good 49223 42223 48646 41646 577 598 1553903 15170 1000 100 1 1 1 1 BMPS He 1104 3278 13837 Good 50954 491293 50353 48528 60m 66 600725 16011 1000 1006 f 12 11 121 BMPS Hs 6101 3473 13837 Good 50834 49828 50238 49230 598 616 7608 20 16448 1000 100 13 11 13 1 BMP8 He 99948 3679 13837 Good 51092 49703 50478 49089 614 622 7071 20 17348 1000 100 4 11141 BMPRIB Hs87223 387913837 Good 50767 49340 5054 48727 613 619 680031 15367 1000 100C 15 11 181 BTC Hs 73105 4079 13837 Good 50398 49164 49811 48579 585 598 579756 15403 1000 100C 16 1 1 16 1 CALML3 Hs239800 4279 13837 Good 49887 48117 49274 47504 613 639 447159 18170 1000 100C 17 X1 171 CEBPA He 76171 4473 13837 Good 48533 47574 47933 46974 600 592 411147 15838 1000 100C 18 11 18 1 CEBPB Hs39029 4679 13837 Good 47516 47333 46949 46766 567 600 328119 17344 1000 100C 19 1 1 191 COLISAT Hs 78409 4879 13837 Good 47939 47614 47369 47044 570 609 322812 17077 1000 100C 20 11 201 CIGF Hs 75511 5079 13837 Good 47772 469682 47158 46348 614 2649 298 33 944410 100 106 f a X1 211 DBH He 2301 5279 13837 Good 47801 48276 47177 47652 624 639 302069 198587 1000 100 22 11 221 DBP Hs 155402 5479 13837 Good 49260 48902 48671 48313 589 629 213724 18912 1000 100C 23 11 23 1 DTR Hs 799 5679 13837 Good 50495 50429 49894 49828 601 613 2818 35 216 26 100 0 100 0 24 V1 241 E21 Hs 96055 5879 13837 Good 50547 49600 49947 49000 600 621 351579 25S 1000 106 f 25 125 1 EF2
22. 617 350 9263 A software application program running on a computer workstation that provides the GUI and allows the user tocalibrate configure control and monitor the instrument The client software communicates with the instrument software to send configuration and control commands to and receive status and configuration information about the instrument A configuration file is a text file that specifies certain system parameters Cable used for the direct connection of two workstation Network Interface Cards NICs NICs have dedicated send and receive lines A crossover cable connects from the send line of one NIC to the receive line of the other NIC Daisy chain is a serial connection of several devices Ethernet is the most widely installed Local Area Network LAN A LAN is a network of interconnected workstations sharing the resources of a single processor or server within a relatively small geographic area i e an office building Ethernet is a set of hardware and signaling standards for used for LANs The most commonly installed systems are 10 100 BaseT A facility network is the network at the facility or institution where you work that links all of the workstations and hubs together Cable made of strands of thin fibers glass or plastic wire that transmits pulses of light Optical fiber carries much more information than conventional copper wire and is in general not subject to electromagnetic interference In ScanArray
23. A Quantitation Protocol uses pre defined settings that can be named saved and recalled to use again Using a quantitation protocol is simple once it is set up just obtain your images select the protocol and start Use a Quantitation Protocol if You have a variety of microarrays You use more than two fluorophores If the printing of the slides does not vary from microarray to microarray e You have complex and advanced settings that you want to use once the settings are set up they can be saved and easily run again 1 5 3 Automatically Quantitating after Scanning When running a Scan Protocol you can select to automatically quantitate after the scan While setting up to run you must select a quantitation protocol to run so the quantitation protocol must already be defined For more information see Running a Scan Protocol in Chapter 4 You also can fully automate your scanning and quantitation using Protocol Groups or Batch Sets For more information see Chapter 6 User Manual pbtsupport perkinelmer com 1 7 Chapter 1 Introduction 1 6 Preparing to Scan To perform a scan the instrument and the workstation must be connected and the instrument must have completed all start up diagnostic tests Start the ScanArray Express and warm up the lasers for 15 minutes and load the microarray or cassette Note Lasers can vary when first turned on After one or two minutes they become J approximately 60 stable
24. Express fiberoptic cable is used to deliver excitation light from the external blue laser to the instrument A dye used to mark DNA It emits light at a given wavelength when excited The scanner needs to know its excitation wavelength and emission wavelength to perform a scan File Transfer Protocol a standard Internet protocol is the simplest way to exchange files between computers It is used to upload and or download files from your workstation to the instrument A gateway is a network point that acts as an entrance to another network It is used to control traffic on your facilty s network A gateway address is the address of a particular gateway on a network A test sample made of photoresist printed on glass it is supplied with a new system When scanned it displays geometric images on your workstation monitor It is used to test image geometry jitter and basic function See Graphical User Interface The portion of an application program that provides a visual interface between the program and the user A GUI consists of windows dialog boxes icons buttons etc that allow a user to control the execution of the program and view its status An initialization process for motion control devices where the motion control device is driven to one particular position to calibrate the control mechanism The output of a scan of one fluorophore ScanArray Express Term Definition Image information Image autosave
25. He 121487 6079 13837 Good 50226 49130 49832 48538 594 619 457030 19849 1000 100C 2 1 21 E23 Hs 1189 6279 13837 Good 49577 48783 48945 48151 632 664 420743 25846 1000 100C N 2 1271 EFA Hs 108371 6489 13837 Good 47868 39817 47323 39072 545 583 1617830 18524 1000 100 28 11 28 1 E25 Hs 2331 6579 13837 Good 49598 48611 48981 47994 617 639 628814 23362 1000 100C 23 11 231 EBAF He 25195 6889 13847 Good 46025 41389 46246 40810 579 603 135229 16636 1000 100C 30 11 30 1 ECGFI Hs739 46 7089 13847 Good 47085 41815 46495 41225 590 610 1289992 19818 1000 100 31 V1 31 EGF Hs 2230 7283 13647 Good 47505 42410 46945 41850 560 560 1244902 13842 1000 1000 32 11 321 EGLI Hs55173 7489 13847 Good A7573 42270 47015 417086 564 577 1087787 14293 1000 106 33 11 1 2 EGFL2 Hs 57652 1268 14037 Good 459681 39698 45322 39059 639 6 amp 0 1370246 16536 1000 100C 34 VW 2 2 EGFL3 He 56186 1489 14037 Good 46887 39731 46283 39127 604 644 1999507 18250 1000 100 35 WwW 3 2 EGFL4 Hs 158200 1663 14037 Good 48571 41122 479684 40515 607 623 1389542 16022 1000 100 36 Vw 4 2 EGFLS Hs 5599 1869 14037 Good 49152 41490 48540 40878 612 636 13700 78 179 65 100 0 100 0 7 Ww 5 2 EGFR Hs 77432 2069 14037 Good 47769 41338 47116 40685 653 662 1382411 17034 1000 100C 38 1 6 2 EGRI Hs 738 2269 14037 Good 46159 40301 45542 39884 617 641 1304978 14234 100 100C 33 WW 7 2 EGR4 Hs 3052 2469 14037 Good 46645 40738 46052 40143 593 624 1248259 15994 1000 100C 40 11 8 2 ENG Hs 76753 2879 14037 Good 4
26. Quality Measurement Formulas PBTsupport perkinelmer com Page viii Preface Preface Conventions Used in this Manual The ScanArray Express uses the Windows 2000 or Windows XP Operating System We assume that the operator is acquainted with the general use of the operating system and therefore provide only an overview of using Windows programs The names of the buttons and their locations are bold The name of windows and dialog boxes are in italics For example e In the ScanArray Express Main Window click Scan to start a scanning procedure All user defined file names for example results files must be Windows compatible i e they cannot contain a slash a backslash a dot or any other Windows reserved character The Cancel button in all windows exits the window you are working in without saving any changes you may have entered Where to Get Help If you need help installing or operating the ScanArray system you can contact PerkinElmer in the following ways Web Site http www perkinelmer com microarray e Customer Service by telephone Tel 617 350 9263 or 800 551 2121 Fax 617 482 1380 E mail PBTsupport perkinelmer com Please be prepared with the following when you call serial number of your instrument version number of the application software from Help About on the ScanArray Express software Main Window nature of the problem e steps you have taken to correct the problem
27. This chapter describes how to scan including how to adjust the display of the images and how to quantitate including reviewing the results using Easy Scan and Easy Quant by Tip To become familiar with how scanning and quantitation work first use the Geometric Test Slide that ships with the ScanArray Express Run an Easy Scan and Easy Quant on the Geometric Test Slide using recommended settings See Chapter 2 Getting Started 3 2 Using Easy Scan AN Note You can stop the scan at any time by clicking Stop on the Main Window The image of what was scanned to that point can be saved just as a fully scanned image can be saved See Saving or Printing Images on page 3 7 User Manual When scanning with Easy Scan you can specify the PMT settings in real time when you are ready to scan If you don t change the settings the settings remain the same as the last time a scan was performed pbtsupport perkinelmer com 3 1 Chapter 3 Using Easy Scan and Easy Quant To run an Easy Scan 1 Load your microarray 2 In the Main Window click Scan The Scan window opens select Run Easy Scan Scan types on systems with an autoloader Scan Area Bun a batch set C Run a scan protocol C Run a protocol group Scan resolution um 95 Mi O20 we 5 Autoloader slot from which to load 1 20 all PMT Laser Fluorophore Gain Power Use Cy5 59 30 Ei Fields for Easy Scan settings
28. Using the Adaptive Threshold method the initial spot mask and background mask are constructed in the same manner as in the Fixed Circle method but this method will quantitate low intensity spots better The Adaptive Threshold is refined pixel by pixel using a modified statistical testing process Mann Whitney test During the test process an adaptive threshold is determined by comparing eight 8 sample pixels inside the spot mask with the eight 8 median background pixels in the background mask The process is repeated with successively brighter pixels in the spot sample until a statistically significant difference is found between the background pixels and the spot pixels The main advantage of the Adaptive threshold method is that it can identify all spot pixels adaptively and can compensate for any morphological change in spots i e if a spot is not exactly round A statistical algorithm is used to define the spot The disadvantage is that background quantitation may become unstable if a large maximum spot diameter is set to compensate for spot size variation To use the adaptive threshold method 1 Select Adaptive threshold and click Set Adaptive Threshold Options Adaptive Threshold Options x Diameters Maximum spot diameter pm Inner background diameter pum Outer background diameter um Spot Identification pValue goo C oon 0005 0 00 C 0025 C 9 050 C 0 100 Calculate Default Values l 2 R
29. a single color User Manual pbtsupport perkinelmer com 3 5 Chapter 3 Using Easy Scan and Easy Quant 3 Click OK 3 2 2 1 Changing the Control Image or Source Experiment Image The first image scanned becomes the Control Image the image to which quantitation compares the other images The Source image is the one that is combined to form the Composite You can change which image is used for the Control or if you have three or more images which is used for the Source experiment To change the control image 1 Onthe Main Window click File then Set Control The Set Control Image window opens Set Control Image The control image is the image to which quantitation compares the other images Please select the image to use as the control image Fluorophore File Name Bbc0032 H omexchange S amples GeoS ample NB beDO32 H omexchange S amples GeoS ample32x32 oF tif When you click OK the image you select will appear on the first tab after the composite on the main screen 2 Select the image to use as the Control Image and click OK The selected image appears in the first tab after the Composite tab in the Main Window To change the source image 1 On the display click Source in the lower right corner The Select a Source Image window opens Select a Source Image The composite image is formed by combining the control image the first image after the composite with another image Please select the image to
30. and unload substrates out of a cassette The cassette holds several substrates 20 in the case of the ScanArray Express HT A set of letters and or numbers encoded into a pattern of bars that can be read and interpreted by a device called a barcode reader Barcodes are most often printed on labels with the labels affixed to objects that need to be identified and tracked plates and substrates in this case Set of scanning protocols for use with an HT Jitter is the deviation in or displacement of some aspect of the pulses in a high frequency digital signal The deviation can be expressed in terms of amplitude phase timing or the width of the signal pulse When the lines and dots in a scanned image appear jagged there is bi directional jitter The amount of allowable jitter is less than 1pixel To adjust jitter contact PerkinElmer Life Sciences Support Microsoft Windows image file format Calibration allows normalization of all ScanArray Express instruments A calibration file is a text file that includes the calibration data of an instrument This file is created at the factory and should not be changed by the user PBTsupport perkinelmer com Term Definition Client Configuration File Crossover Cable Daisy chain Ethernet Facility Network Fiberoptic Cable Fluorophore FTP Gateway Address Geometric Sample Slide GUI Graphical User Interface Homing Image Technical Support 800 551 2121 or
31. change the palette and adjust the display to see the spots better Adjusting the display does not change the data for quantitation or the data in the file only the appearance of the image on the screen You can change the control image and if there are more than two images you can set or change the source experiment image Scan Quantitate Configure amp File File Configure Spot Viewer ite F Mouse is at 6699um 17097m Pixel intensity ratio 0 91 Zoom in Zoom Out Zoom Full Dragging with mouse Draws Circle from Side Laser 2 634 nm Remove Circle Not Installed Full Slide Laser 4 488 nm Not Installed Laser 5 514 nm Not Installed Autoloader iin oor Not Installed Cassette Load Status Not Installed Palette Source j 4000 Full color threshold 53 7K Loaded microarray 3x3 Median Restore None Palette Remove 3x3 Median Filter Auto Adjust Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Quant Chapter 3 To adjust the display 1 The ScanArray Express scans the microarray using the Auto Adjust settings that provide the best contrast between the spots and the background to make the spots more visible Click Unadjusted to remove the auto adjustment and display the raw data 2 Click the 3x3 Median filter to make the image appear smoother and less noisy cleaning up miscellaneous pi
32. combine with the control image Fluorophore File Hi ame Cy5 BbcO0032 Homexchange S amples If you wish to set the control image instead click File on the main window and then click Set Control Cancel 2 Select the image to use as the Source image the selected image appears in the first tab after the Control image tab Note With the images still displayed you can immediately quantitate See Using Easy Quant on page 3 8 3 6 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Quant Chapter 3 3 2 3 Saving or Printing Images If you did not select to save images automatically by checking the Autosave checkbox es before scanning you can save the images after scanning You can also save a portion of an image to make a smaller file To save images that were not automatically saved 1 On the Main Window click File in the Configure amp File group then click Save or Save as to save the selected image Click Save All to save all of the images Open Image Set from Array Informatics Save Save in Save As Array Informatics Save All Save Portion of Image Close View Header Close All Print Set Control Image In the Save Image dialog box that opens enter a filename and pathname for the image To save a portion of an image 1 2 Select an image tab On the Main Window click File in the Configure amp File group then click Save Po
33. commas The major sections of a CSV file are described in Section A 4 1 Each field in the file are described in the Section A 4 2 Description of the File Format Table A 5 lists the major sections of the ScanArray Express CSV file and a brief description of each Table A 5 Description of Major File Sections Section Description Header Contains information about the product and version number and the Array lnformatics database name if the data is to be saved to Arraylnformatics General Contains date instrument information user log in name and other Quantitation Contains values for the quantitation method selected Parameters Quality Measurement Critieria Arary Pattern Information Image Information Normalization Information Data Contains the values for quality measurement Contains descriptive information about the spot size spacing and array layout Contains information about the image for example the fluorophore the barcode etc Contains values for the normalization method The first row contains the labels and the following rows are the measurements one for each spot There may be up to four Channels Note Chx in the following columns represents Channels x x 1 2 3 4 A 4 2 Description of the File Fields User Manual The following is a description of each field in the CSV file BEGIN HEADER PerkinElmer Life Sciences name of the company ScanArrayCSVFileFormat 2
34. diagram on the left side of the window is the full slide area showing the selected scan area outlined in a white box Select or change a scan area by using the mouse cursor to outline the desired area on the diagram Alternatively you can enter specific X and Y coordinates or select the full microarray as described under Area Co ordinates below You can enter precise coordinates here for the scan area instead of selecting an area with the mouse Start position X mm Area Width mm Start position Y mm Area Height mm Click here to set or reset the scan area to the full microarray Click here to open the Scan Area for Zoom window where you can zoom in on the display and get help with selecting an area pbtsupport perkinelmer com 3 3 Chapter 3 Item Using Easy Scan and Easy Quant Description Automatically save images Automatically save in Array lnformatics Check this box to automatically save the scanned images locally When you click Start a dialog box opens where you specify a filename and path for saving the image s Check this box to automatically save the scanned images in the Arraylnformatics database If your scanner is not integrated with Arraylnformatics this field is dimmed and unavailable to select 3 2 2 Adjusting the Display 3 4 The scanned image s display on the Main Window a tab for each fluorophore and a Composite tab of both fluorophores if two were selected You can
35. for Easy Scan Focus position jm Laser power Laser Settings IV Allow user to skip laser warm up period Array Informatics Other ro 30 2 Refer to the following table Item Description Allow user to skip laser warm up period Settings for Easy Scan Focus Position lum Laser Power Check this box to allow the user to start scanning before the lasers are warmed up 5 or 15 minutes depending on the type of laser This does not cause any damage but the sensitivity of the resulting scans will vary If the user is concerned with repeatability or uniformity of scanning they should wait for the lasers to warm up This defines the settings of the focus position and laser power for all Easy Scans These settings are not changeable from the Scan window The only time the focus position needs to be changed is when the user changes to a different type of slide substrate such as a gel or membrane slide where the imaging surface is at a different height than it is with a glass side The default is 0 zero Valid values range from 150 to 2000 um The default is 90 percent Valid values range from 0 to 100 percent User Manual pbtsupport perkinelmer com 7 3 Chapter 7 7 2 3 Quantitation Settings System Settings The quantitation settings let you set some default system settings for quantitation To change quantitation settings 1 Click the Quantitation tab Application Se
36. for Measurement Control and Laboratory Use Europe CRDH Title 21 CFR 1040 10 Class Laser Product USA EN 60825 1 1994 Laser Equipment Classification Requirements and Users Guide for Laser Safety IEC 825 Class Laser Product FCC Part 15 Class A Radiated and Conducted USA EN55011 1991 Class A Radiated and Conducted Europe ICES 003 Industry Canada Interference Causing Equipment Standard Digital Apparatus Class A EN50082 1 Electromagnetic Compatibility Generic Immunity Standard Part 1 Residential Commercial and Light Industry EN61000 4 2 Electrostatic Discharge EN61000 4 3 Radiated Electromagnetic Fields EN61000 4 4 Electrical Fast Transient Burst EN61000 4 5 Surge Immunity Requirements EN61000 4 6 Conducted Disturbances Induced By Radio Frequency Fields EN61000 4 11 Voltage Dips Short Interruptions and Voltage Variations Immunity Tests This device complies with Part 15 of the FCC Rules Operation is subject to the following two conditions 1 This device may not cause harmful interference and 2 This device must accept any interference received including interference that may cause undesired operation This class A digital apparatus meets all requirements of the Canadian Interference Causing Regulations Cet appareil num rique de la classe A respecte toutes les exigences du R glement sur le mat riel brouiller du Canada ScanArray Express System Specifications Appendix C Sc
37. for each Channel if there are 2 or more channels quantitated Chx N Median normalized median Chx N Mean Chx N Median B Chx N Mean B Following Columns Repeated for each Non Control Channel Chx N Ratio of Medians Chx N Ratio of Means Chx N Median of Ratios Chx N Mean of Ratios Chx N Rgn Ratio Chx N Log Ratio END DATA User Manual pbtsupport perkinelmer com Page A 13 Appendix Supported File Formats A 5 _ TIF Files The TIFF TIF file format Tag ged Image File Format is an industry standard 16 bit file format for storing images The TIF files can be transferred between different platforms A 14 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Declaration of Conformity Aopen B This device conforms to the requirements of US 21CFR1040 10 and 1040 11 at date of manufacture This instrument is designed and certified to meet the following regulatory and safety standards B 1 Electrical and Mechanical Safety Standards The product is UL listed USA and Canada to the following standard UL 3101 1 1993 Electrical Equipment for Laboratory Use USA amp Canada The product is T V certified to the following standard e EN 61010 1 A2 1995 Safety Requirements for Electrical Equipment for Measurement Control and Laboratory Use Europe B 2 Laser Safety Standards CRDH Title 21 CFR 1040 10 Class I Laser Product USA EN 60825 1 1994 Laser Equipment Classification R
38. for the calculation of the background statistics Enter the maximum percentile value for the calculation of the background statistics 3 Click OK 5 4 6 Normalization Methods User Manual Normalization corrects the intensity of each spot for variations in the overall intensity of the images with respect to the control image e The LOWESS method Locally Weighted Scatter Plot Smoothing carries out robust locally weighted scatter plot smoothing for both equally spaced and non equally spaced data For more information on the LOWESS method refer to the following paper Y H Yang S Dudoit P Luu and T P Speed Normalization for cDNA Microarray Data SPIE BiOS 2001 San Jose California January 2001 The paper can be found at the following web address http stat www berkeley edu users terry zarray Html normspie html e The Total method uses the ratio of total of all spot pixels pbtsupport perkinelmer com 5 17 Chapter 5 Quantitating with Protocols 5 4 7 5 5 Quantitation Protocol Summary The Summary lists all of the parameters you have selected for quantitation and their values To view a summary of the protocol settings 1 Open the Summary window where you can review the settings for this protocol Quantitation Protocol x Composite Cy3 Control Cy5 1 Basic Information 2 Image Registration Zoom In Zoom Gut Zoom Full 5 Summary These are the parameters you have entered
39. images 1 Click the tab of the image you want to look at to bring it to the front 2 Use the mouse to zoom in on the details or to shift the view to a different part of the microarray e A mouse click on either the full slide or large image centers the field of view on the point where you click the mouse To shift the view to a completely different part of the microarray click a point on the Full Slide To shift the view slightly click a point in the large image display e To zoom in or zoom out on the display click the appropriate Zoom button located above the full slide view The white rectangle in the Full Slide shows the size of the field displayed in the Main Window and it changes size with the zoom level 2 4 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Getting Started Scan and Quantitate the Geometric Test Slide Chapter 2 3 To view the average intensity of a certain area click the Draw Circle from the Center Side button This enables you to draw a circle around the desired area using the mouse The average intensity of spots within the circle is displayed Run Composite Cy3 Control Cy5 Log Diagnostics Help Scan Wt Zoomin Zoom Out Zoom Full Quantitate Configure amp File File Configure Laser 4 488 nm Not treed Laser 5 514 rnm Not Installed Autoloader joor Not Installed Cassette Load Status Not Installed Artaylniomatics erven Not Installed
40. is not integrated ArrayInformatics is a microarray gene expression database and visualization software available from PerkinElmer If you are working in an integrated laboratory refer to the Microarray Laboratory Integration Guide that shipped with the ArrayInformatics software 1 1 1 6 The User Interface The User Interface the part of the ScanArray Express software that you see displayed on the workstation monitor screen lets you enter information or commands and receive status information The user interface opens to the ScanArray Express Main Window described on the following page pbtsupport perkinelmer com 1 3 Chapter 1 Introduction 1 2 The ScanArray Express Main Window When you start the ScanArray Express the user interface displays the Main Window on the monitor screen as shown in Figure 1 2 below This is your starting point for sending commands entering information or receiving status information This is also where scanning images and quantitation results display Note Some ScanArray Express systems are for quantitation only if you are working on a Quantitation system you will not see the Scan button or associated windows Run Configure amp File Buttons Click Scan to select a scanning type and begin scanning Status buttons Display the status of Click Quantitate to select a quantitation type and begin quantitating the lasers and other instrument hardware Turn lasers on or off Imag
41. lt lt lt lt lt lt lt lt lt K Fluorophores beginning with cannot be scanned with current hardware Figure 7 2 List of Fluorophores Window 2 Sort the list optional by Name Description Excitation Peak or Emission Peak 3 Click one of the following in the List of Fluorophores window Add to create a new fluorophore Change after selecting a fluorophore to modify an existing one Duplicate makes a duplicate of the highlighted fluorophore to help you create a new fluorophore from one that is already close to what you want e Delete after selecting a fluorophore to delete it User Manual pbtsupport perkinelmer com 7 7 Chapter 7 System Settings View Usage after selecting a fluorophore to display a list of protocols which include the selected fluorophore You cannot delete a fluorophore that is being used by a protocol 4 Click Add The Fluorophore dialog box displays Fluorophore Name Description Excitation Peak nm Emission Peak nm E s7 Advanced Options Palette Red Use the palette chosen above For all images of this fluorophore For composite images only use the rainbow palette for simple images a core Figure 7 3 Fluorophore Dialog Box 5 Enter the name and a brief description of the fluorophore in the appropriate fields 6 Enter the Excitation Peak and Emission Peak of the fluorophore These values can be obtained from the
42. of spots in all subarrays Completely Reset Template Modify Template Using Changes Only This describes the position and size of each spot in a subarray Enter the number of rows and the number of columns of spots that are in each subarray Enter the horizontal and vertical spot spacing measuring from the center of a spot to the center of the spot in the next row or column Enter the spot diameter in um This forces the spots into alignment for quantitation The straightening does not show on the displayed image but affects the quantitation results This resets the template Modifies the template using only the changes just entered 3 3 1 2 Adjusting the Template and Registering Images After loading the template adjust it to precisely fit the subarrays and register the images The images are not perfectly aligned if traces of individual color such as green and red are visible around the outside of each spot Use the Zoom controls as needed to make it easier to adjust the template and view the registration To adjust the template 1 Click Adjust Template and Register Images The Adjust Template and Register Image window opens see Figure 3 4 on the next page Technical Support 800 551 2121 or 617 350 9263 ScanArray 1M Express Chapter 3 Using Easy Scan and Easy Quant Adjust Template controls Register Image controls User Manual Adjust Template and Register Images Adjust Template t 93 Zoomi
43. save images in Arraylnformatics the rectangle edges with the mouse or use the the box labelled Area Co ordinates at Cancel right 7 Use the following settings appropriate for the Geometric Test Slide e Set the Scan Resolution to 10 um e Ifyou are using an HT instrument indicate the cassette slot into which you placed the slide Select Cy3 as the First Fluorophore with PMT Gain at 55 this will be the control image To change the fluorophore click the button labelled with the fluorophore name select the appropriate fluorophore from the list and click OK Select Cy5 as the Second Fluorophore with PMT Gain at 65 this will be the experimental source image 2 2 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Getting Started Scan and Quantitate the Geometric Test Slide Chapter 2 Set the Scan Area by entering the following values into the Area Co Ordinates edit boxes Item Setting for GeoSlide Start position X mm 0 75 Area width mm 7 0 Start position Y mm 14 00 Area height mm 7 5 8 Click Start The ScanArray Express acquires a pair of images from the Geometric Test Slide using the Cy3 and Cy5 fluorophores and automatically ejects the microarray when finished 2 3 Viewing the Images The scanned images display in the Main Window each with its own tab Cy3 Cy5 and the Composite of the two fluorophores You can click an image tab to bring that image to t
44. t Hs 6540 5079 14047 Good 40926 46310 40302 624 888 1413394 160737 1000 100C 53 11 He 248110 5278 14047 Good 4gi07 41721 47484 M098 623 16727 1000 100i aie Loaded Spies microarray Gene name Show Hide Show Hide SetQualiy Set View ACVAI Spote by Statue Columns Citeia SpotStatus Spot Pisete None 2 5 1 Spreadsheet Each row in the spreadsheet is the data from one spot including the gene names and ID numbers that were imported from the GAL file You can scroll vertically to see the data for each spot You can scroll horizontally to view the 55 columns of data for any spot or click Show Hide Columns to hide any columns you don t want to see The spreadsheet is interactive click on a row in the spreadsheet and see the spots that created that data in the spot viewer on the left side and also see those spots highlighted and centered in the full images by selecting the Cy3 Cy5 or Composite image tab Click on a column to sort using that column Note If you have Microsoft Excel on your system you can view the spreadsheet in Excel by clicking View as Excel Spreadsheet in the bottom right corner of the Spreadsheet tab User Manual pbtsupport perkinelmer com 2 9 Chapter 2 Getting Started Scan and Quantitate the The following buttons on the Spreadsheet tab let you set the spot status and select which data to view They are described fully in Viewing the Spreadsheet in Chapter 3 Button D
45. that describe the completed protocol Parameter Value No of subarrays rows 1 No of subarrays cols 1 Rotation of template 0 00 Pin spacing H mm 450 Pin spacing V mm 450 Rows of spots per subartay 32 Cols of spots per subarray 32 Spot spacing H um 200 Spot spacing V um 200 Spot diameter um 100 Subarray 0 0 top left comer x am 6668 top left comer y urn 16986 Quantitation method Fixed Circle Signal range low 0 Signal range high 35 Bkand range low 5 Bkgnd range high Bkgnd diam inner um 0 Bkgnd diam outer um 0 Figure 5 6 Quantitation Protocol Summary Window 2 Click Finish to save your protocol Creating a Spreadsheet Autosave Protocol Spreadsheet Autosave Protocols specify the filenaming conventions file type and file location to use when automatically saving the quantitation results Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Quantitating with Protocols Chapter 5 To create a spreadsheet autosave protocol 1 On the Main Window click Configure in the Configure amp File group In the Configure menu that displays click Spreadsheet Autosave Protocol The List of Spreadsheet Autosave Protocols window opens List of Spreadsheet Profiles Description Pattern Sort By Name Description Path D 2 Sort the list optional by Name Description or Path 3 Click one of the following in the List of Sp
46. the database 7 12 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Settings Chapter 7 7 5 1 Backing up the Database You should have a set procedure for when to back up the database and how to name the backup file so that in the event you need to restore the database the correct backup file is easily identified To back up the database 1 Click Back Up Database 2 Use the Browse window to select a folder A default name for the backup is supplied and it is recommended to use the default name 7 5 2 Restoring the Database Restore the database only if absolutely necessary that is if the file has been lost or accidentally deleted or if you have accidentally deleted a protocol from a previous software version for example from v1 1 when running v2 0 the A Note Avoid restoring from previous versions of the software If you do restore a backup ScanArray Express software will have to be reinstalled To restore the database 1 Click Restore Database 2 Use the Browse window to find the backup file that was named and saved during backup User Manual pbtsupport perkinelmer com 7 13 Chapter 7 7 6 Service Features System Settings Service features are used for calibration and configuration of the instrument and for controlling the instrument software operation Certain service features are intended for use by authorized personnel only or under the direction of service or tec
47. the previous window the Sensitivity Calibration Area window To run Automatic Sensitivity Calibration 1 Open the Scan Protocol Automatic Sensitivity Calibration window Scan Protocol Automatic Sensitivity Calibration Average spot size pm 100 Target signal intensity 90 Sensitivity adjustment method 5 Sensitivity Calibration Keep laser power fixed vary PMT gain Keep PMT gain fixed vary laser power ec 6 Tools Finish Cancel Figure 4 10 Scan Protocol Automatic Sensitivity Calibration Window 2 Use the following table for information on settings Item Description Automatically Check this box to have AutoSensitivity run automatically on every scan calibrate sensitivity with this protocol for each microarray Average spot size Enter the average diameter of your microarray spots for the greatest um particle dust suppression If the spots are poorly formed set the average spot size to a smaller value for better results The minimum and maximum spot sizes are 75 and 500 um The default spot size is 100 um User Manual pbtsupport perkinelmer com 4 13 Chapter 4 Scanning with Protocols Item Description Target signal intensity AutoSensitivity automatically adjusts the laser or PMT so the brightest feature in the Sensitivity Calibration area is at this percentage of full scale The Target signal intensity for poorly formed or mottled spots should be lowered to avoid saturation
48. to automatically save the scan images and or Arraylnformatics quantitation data from this range of slots in the Arraylnformatics Database database Remove the Check this box to easily remove the quantitation protocol and the quantitation protocol spreadsheet autosave protocol from all of the slots in the range that you from all slots in the are configuring range User Manual pbtsupport perkinelmer com 6 11 Chapter 6 Item Automating Scans and Quantitation Description Set protocol group Set scan protocol Set image autosave protocol Set Quantitation protocol Set spreadsheet autosave protocol Check this box to select one or more protocol groups to run on the range of slots Checking this box enables the Protocol Group box where any configured protocol groups will display Check this box to select one or more scan protocols to run on the range of slots Checking this box enables the Scan protocol box where any configured scan protocols will display Check this box to select an image autosave protocol to save the scanned images automatically Checking this box enables the Image autosave box where any configured protocols will display Check this box to select one or more quantitation protocols to run on the range of slots Checking this box enables the Quantitation protocol box where any configured protocols will display Check this box to select a spreadsheet autosave protocol to save the quantitation res
49. um Spot diameter pm 100 I Straighten rows and columns of spots in all subarrays Completely Reset Modify Template ance Template Using Changes Only Figure 3 3 Define Quantitation Template Dialog Box 2 Provide the subarray and spot information as described in the following table Item Description Subarrays Number of rows and number of columns of subarrays Rotation Pin spacing horizontal Pin spacing vertical Straighten rows and columns of subarrays Describe the geometry of the microarray Enter the number of subarray rows and the number of subarray columns If the microarray is skewed on the substrate you can rotate the template to align the template to the microarray Enter the horizontal and vertical pin spacing that was used to create the microarray 4 5 if 384 well plates were used to print the microarray 9 0 if 96 well plates were used to print the microarray Custom to enable the edit boxes where you can enter values for custom pin spacing This forces the subarrays into straight rows and columns for quantitation The straightening does not display on the image but affects the quantitation results User Manual pbtsupport perkinelmer com 3 11 Chapter 3 Item Using Easy Scan and Easy Quant Description Spots Rows of spots and Columns of spots per subarray Horizontal spot spacing center to center um Spot diameter Straighten rows and columns
50. within the brightest spots The minimum and maximum target intensities are 50 and 100 The default target intensity is 90 Sensitivity adjustment For the best signal to noise ratio go to Fluorophores and set the laser method power to 95 for all fluorophores then choose Keep laser power fixed vary PMT gain 3 Click Finish to save the Scan Protocol 4 4 6 Scan Protocol Tools The Scan Protocol Tools window provides tools you can use to determine or refine correct settings for this protocol After you run a tool and accept the results the settings are automatically saved back to the protocol When you save the protocol all settings are saved A Note The Quick Scan can be used to test protocol parameters to see the resulting scanned image Only experienced users should use the other scan protocol tools All of the tools perform specialized scans and you must have a microarray in the scanner before you run them Tools include e Quick Scan which allows you to adjust the settings during a scan and save them e Line Scan to help you determine the appropriate focus position laser power and PMT Gain for various fluorophores Automatic Sensitivity Calibration for determining appropriate laser power and PMT Gain settings and e Automatic Focus Calibration for determining the appropriate focus setting When a tool has finished running click Accept the Changes to save the information to the protocol
51. 0 name and version of the file format ScanArray Express 2 0 name and version of the software pbtsupport perkinelmer com Page A 9 Appendix Supported File Formats AIDBServerName SomeName Added for AIDB this row is available only if the data was intended for AIDB Number of Columns 62 Actual Number of columns here number of Data columns number of data records END HEADER BEGIN GENERAL INFO DateTime 12 12 01 8 00 Date and time when the image was acquired in 24 hour format GalFile c images test gal gal file which associates names and ids Scanner type of scanner used Model and serial number User Name Windows Logged in user name Computer Name Computer Name Protocol _ Scan Protocol used to scan the images Quantification Method Quantiation method Quality Confidence Calculation Quality criteria user User comments aser comments Image Orgin 0 0 image origin relative to the scan area Temperature temperature of the scanner in volts Laser Powers 10 10 Naser power Laser On Time Laser on time for each laser in minutes PMT Voltages 10 12 voltages InstrumentID Added for AIDB this is the AIDB instrument token returned by AIDB present only if the data is intended for AIDB END GENERAL INFO BEGIN QUANTIATION PARAMETERS Values in this section depend on the quantitation method selected For Adaptive Circle Express method Minimum Percentil value Maximum Percentile value Fo
52. 0C 16 11 He 239600 4273 13837 Good ABZ 49274 47504 613 639 447159 18170 1000 10C 17 11 He 76171 4473 13837 Good A7574 47933 46974 600 592 411147 15838 1000 100C Ratio 0 83 18 11 Hs 99029 4679 13837 Good 47333 46949 46786 567 600 328119 17344 1000 100i 5 13 4 He 78409 4873 13837 Good A7614 47369 A7044 570 609 322612 17077 1000 10C 20 11 Hs 75511 5079 13837 Good 46962 47158 46348 Gl4 2649 269833 944410 1000 100C a 4 Hs 2301 5278 13837 Good 48276 47177 47652 624 639 302069 19587 1000 100k Autoloader 22 11 Hs 155402 5479 13837 Good 48902 48671 483 569 629 213724 18912 1000 100C Door 23 ii Hs 799 5679 13837 Good 50429 49894 49828 60 613 289835 21628 1000 100C Not Installed 24 1 1 Hs 96055 5879 13837 Good 49800 49947 49000 600 621 351579 22551 1000 100C i me 2 3 Hs 121487 8073 13837 Good 43130 49632 48536 594 619 457030 19849 1000 100C eee 25 Ww Hs 1189 6273 13837 Good 48783 48945 48151 632 664 4207 43 25846 1000 100C Not aad Neder 47200 27 Ww Hs 108371 6489 13837 Good 39517 47323 39072 545 563 1617830 18524 1000 100C 3 28 i Hs 2331 8573 13837 Good 49511 48981 47994 617 639 628814 23362 1000 100C 29 WW Hs 25195 6889 13847 Good 41389 46246 40810 579 603 135239 16636 1000 100C 30 11 Hs 73946 7089 13847 Good 4815 46495 41225 590 610 1289992 13818 1000 100C 31 11 Hs 2230 7289 13847 Good 4240 46945 41850 560 560 1244902 13842 1000 100C 32 11 Hs 55173 7489 13847 Good 42270 4705 41706 564 577 1087787 14293 1000 10C 33 11 Hs 57652 1269 14037 Good 39
53. 2 Viewing the Quantitation Results 0 cece eens 3 14 Viewing the Spreadsheet soncdadseawodaneemetrs o Keeani dee 3 15 Filtering the Spreadsheet Data 0 000 ee 3 16 Viewing the Scatter Plot 2 244 94 ove vans ae aadwk eee aaa s 3 20 Viewing the Distribution Plot 0 4 p08 5 0 44 aana 44 ennme aes tes 3 20 Viewing Information About the Images 0 00000 e eee 3 22 Saving the Quantitation Results ota ees eee de ieee et xe 3 23 Chapter 4 Scanning with Protocols OVV EW 553 aa Sie e GAA ERIS We RSS hes Wee a Re 4 Running a Scan Protocol 4 0 v2 8 Ane Se ete eee ARG h eek eed hes 4 Other Scan Settings ght ete geal MN di i Dae wee Nee aid GN 4 3 Automatic Quantitation after Scanning 0 0 ces 4 4 Creating a Scan Protocol 25 c ccc04 4 estekasa Med dihed aye d Lhihas paomadaues HE wSO 4 4 The Scan Protocol Wizard oc 2 se ealgarda ice Hawai wala late Rasa 8s awe 4 5 Basic Infofmation rese a wea Gee eae E Baud 4 6 Selecting the Scan Area srs citer acwqa LEN GAS AS oa OOS CARE I OE 6 vores ed 4 7 Fl orophores 5 lt wind seed eRe eae E ETE ead Bae ee BN RE OA 4 9 Sensitivity Calibration Areas 4 ag 4cnut gee swesG a4 4a seuohagya sn 4 10 Automatic Sensitivity Calibration 0 0 00 ce eee 4 13 Scam Protocol To0olsi034 0 te pagu eds whee acta ee Re we RE 4 14 Using Scan Protocol Tools tases 3 ene g wears 6 oS 2d 5 ee SS eae Ve ee 4 15 Running the Quick Sans 230 0 3 Peace
54. 3 Distribution Plot Chapter 2 The Distribution Plot allows you to see trends that are area sensitive on the slide for example if one side of the slide is being over or under washed or if a pin is partially clogged Spot Index 785 Gene name 2 Distribution Plot of Cy Ratio of Mean Spat fowm_J 2 Zoomin Zoom Out Zoom Full Sia i anh You can select a column of spots to plot from any one of the images or any images with the control image Select a Column to Plot Category Column General Cy5 Control Cy3 User Manual Ratio of Median Spot Ratio of Mean Spot Median of Pixel Ratios Mean of Pixel Ratios Regression of Pixel Ratios M Log2 Ratio of Median Spot M Log2 Ratio of Mean Spot A Mean of Log2 Median Spot 4 Mean of Log2 Mean Spot Ratio of Median Norm Spot Ratio of Mean Norm Spot Median of Norm Pixel Ratios Mean of Norm Pixel Ratios Regression of Norm Pixel Ratios M Log2 Ratio of Median Norm Spot M Log2 Ratio of Mean Norm Spot 4 amp Mean of Log2 Median Norm Spot A Mean of Log2 Mean Norm Spot Std Dev of Pixel Ratios p for Regression Ratios pbtsupport perkinelmer com 2 11 Chapter 2 Getting Started Scan and Quantitate the 2 6 Saving the Images To save the images 1 Onthe Main Window click File in the Configure amp File group then click Save or Save as to save the selected image Click Save All to sav
55. 33 nm two Emission Filter Wavelengths of 570 nm and 670 nm Technical Support 800 551 2121 or 617 350 9263 ScanArray TEX press Introduction User Manual Chapter 1 1 1 1 3 The ScanArray Express The ScanArray Express can be upgraded to alternative lasers filters barcode reader and autoloader This model supports up to five Laser Excitation Wavelengths of 488 nm 514 nm 543 nm 594 nm 612 nm and 633 nm up to 11 Emission Filter Wavelengths of 508 nm 522 nm 530 nm 549 nm 570 nm 578 nm 592 nm 614 nm 660 nm 670 nm and 694 nm 1 1 1 4 The ScanArray Express HT The ScanArray Express HT includes a built in autoloader and can be upgraded to alternative laser filters and barcode reader This model supports up to five Laser Excitation Wavelengths of 488 nm 514 nm 543 nm 594 nm 612 nm and 633 nm up to 11 Emission Filter Wavelengths of 508 nm 522 nm 530 nm 549 nm 570 nm 578 nm 592 nm 614 nm 660 nm 670 nm and 694 nm 1 1 1 5 The ScanArray Express in an Integrated Microarray Laboratory If your laboratory is integrated with an ArrayInformatics database that resides on a server or workstation on the network you can retrieve information from and send information to the ArrayInformatics database Selection buttons for ArrayInformatics on the ScanArray Express user interface are activated if your ScanArray Express is configured for integration or dimmed and inactive if your ScanArray Express
56. 5 4 3 1 Defining a Template Using a GAL File A GAL file output from the SpotArray includes a gene list and information about each spot on the microarray To define a template using a GAL file 1 In the Quantitation window under Template click From GAL File The Open GAL File dialog box opens Open GAL File Look in samples a Contents gal File name Yeh a Files of type GAL gal 5 8 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Quantitating with Protocols Chapter 5 2 Highlight the desired file and click Open The dialog box closes and the name and path of the selected GAL file displays under the From GAL File button in the Protocol Wizard Template window 5 4 3 2 Defining a Template From Specifications Using specifications you establish the array pattern by entering numerical data that defines the subarrays and spots To define a template from specifications 1 Inthe Protocol Wizard Template window click From Specifications to enter values that define the geometry of the array The Template Specifications window displays Template Specifications Subarrays Number of rows of subarrays Number of columns of subarrays Rotation degrees 0 00 Horizontal pin spacing mm 45 C90 Custom 450 Vertical pin spacing mm 45 90 C Custom 15 I Straighten rows and columns of subarrays Spots Rows of spots per subarray Columns of spots per suba
57. 8660 45059 48060 44459 600 667 926554 36123 1000 1000 a1 11 3 2 EPAS I Hs 168082 2873 14037 Good 49158 46893 48588 46323 570 620 697723 15581 1000 106 f 2 11 102 EPSI5 Hs79095 3079 14037 Good 50054 48333 49490 47769 564 609 732800 18518 1000 106 f e 11 112 EPS8 He 2132 3279 14037 Good 51793 49793 51193 49187 606 635 658110 18140 100 106 f 44 1112 2 ERBB2 Hs 173664 3473 14037 Good 5o58 50203 50255 49600 603 622 539871 15390 1000 100 45 1 1 13 2 ERBB3 Hs 199087 3673 14037 Good 50523 49297 49935 48709 S88 609 556735 17283 1000 100C 4 11 142 ERBB4 Hs1939 3879 14037 Good 50739 48960 50095 48316 644 633 521847 15881 1000 100C a7 V1 18 2 EREG He 115263 4073 14037 Good 48938 48105 48299 47466 639 667 486188 22116 1000 100 4 11 16 2 FGF Hs 75297 427914037 Good 49075 47914 48430 47269 645 764 433834 48323 100 106 f 43 11 17 2 FGO Hs 248049 4479 14037 Good 48023 47384 47460 46821 563 653 418270 19879 1000 100C 50 1 1 18 2 FGFI Hs 249165 4673 14037 Good 48719 48264 48100 47645 619 646 477686 19097 1000 1006 f 51 13 19 2 FGFI2 Hs 124752 4879 14047 Good 47333 41085 46743 40495 590 618 1432263 170 61 100 0 100 1 52 11 20 2 FGFI3 Hs6540 5079 14047 Good 46334 40926 46310 40302 624 888 1413394 160737 1000 100 53 XI 21 2 FGF4 Hs 248110 5279 14047 Good 48107 41721 47484 41098 623 648 1420954 16727 1000 100x 4 ae Loaded Spot Index 1 5 E Gene name Show Hide Show Hide SetQualiy Set View View as ACVRT Spotsby Status Columns Crea SpotStatus
58. 898 45322 39059 639 660 1370246 16536 1000 100C 34 11 Hs 56186 1469 14037 Good 39731 46283 39127 604 644 1399507 18250 1000 100C 35 il Hs 158200 1689 14037 Good 41122 47964 40515 607 623 13695 42 16022 1000 100C 3 inl He 5599 1889 14037 Good 41490 48540 40678 612 636 1370078 179365 1000 100C Madian aAA 7 t1 Hs 77432 2069 14037 Good 41338 47116 40685 653 662 1362411 17094 1000 100C 38 11 He 738 40301 45542 99684617 641 1304978 14234 1000 10C 33 Ww He 3052 40736 46052 40143 593 624 1248259 15994 1000 100C 40 WW Hs 76753 45059 48060 44459 600 667 926554 36123 1000 100C 41 WwW Hs 166082 2879 14037 Good 46893 48588 46323 570 620 697729 15581 1000 106 42 Ww Hs 73095 3073 14037 Good 48333 49490 47769 564 609 732800 18518 1000 100C ry Ww Hs 2132 3273 14037 Good 49793 51193 49187 606 63 6581 10 18140 1000 100 4 Ww Hs 173658 3473 14037 Good 50203 50255 43600 603 622 599871 15390 1000 100i 45 v1 Hs 199067 3679 14037 Good 49297 49935 48709 588 609 5567 35 17283 1000 100i 46 v1 Hs 1939 3879 14037 Good 48960 50095 48316 644 633 521847 15881 1000 100i 7 v1 Hs 115263 4073 14037 Good 48105 48299 47486 639 667 486188 22116 1000 100i 48 4 Hs 75297 4273 14037 Good A7914 48430 47269 645 764 433894 48323 1000 100C 43 Ww Hs 248049 4479 14037 Good 47384 47460 46821 563 653 418270 19873 1000 100C 50 il He 249165 4679 14037 Good 48264 48100 47645 619 646 477686 19097 1000 100C 51 11 Hs 124752 4879 14047 Good 41085 46743 40495 590 618 1432263 17061 1000 100C 52
59. Accept Changes to accept the results Automatic Sensitivity Calibration Laser PMT Fluorophore Power Gain Scanning Pending Progress Cy3 Cy5 4 5 4 Automatic Focus Calibration 4 22 Only experienced users should use this tool The Automatic Focus Calibration tool scans a selected area of a microarray with a single fluorophore and determines the appropriate focus position To perform Automatic Focus Calibration 1 Click Run in the Automatic Focus Calibration area of the Scan Protocol Tools window The Automatic Focus Calibration dialog box displays Automatic Focus Calibration Select the fluorophore to use for the calibration PMT Laser Fluorophore Gain Power y Fluorophore cannot be scanned with current hardware Cancel Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 2 Select the fluorophore to use for the calibration To change the area to be used for focus calibration click Adjust the Focus Line to move to an area with a lot of features The Focus Line Definition window opens where you can move the focus line Focus Line Definition six Help amp Directions Composte G8 08 Zoomin Zoom Out Zoom Full Li wT To Madity the Sean Line line and release the mouse Focus Line Start position mm 00 Start pe LY mmm 3650 Line length mm 2200 Line thickness mnt
60. In the Configure menu click Scan Protocols Configure Configure amp File File Autosave Configure Protocols Protocols Quantitation Spreadsheet Autosave Protocols Quantitation Protocols Automation Protocol Groups Batch Sets Advanced Application Service Settings Features Eluorophores Barcodes Database Maintenance Figure 4 2 Configure amp File group and Configure menu 4 4 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 2 The List of Scan Protocols window opens The ScanArray Express includes several default protocols that display in this window along with any protocols that have been created List of Scan Protocols Description Dr Thomas Dr Thomas Dynamic Repeat Single Cy5 scans geo slide dots Geo Slide 32x32 Geo Slide 32x32 Demo Geometric Geometric Rotated Uniformity Single Cy5 scan geo slide dots Static Repeatabil 4 consecutive Cy5 scans geo sl Protocol cannot be executed with current hardware Resolution um Figure 4 3 List of Scan Protocols Window Modify Add Change Duplicate Delete View Usage Sort By Name Description Resolution 3 Sort the list optional by Name Description or Resolution 4 Click one of the following in the List of Scan Protocols window to open the Scan Protocol wizard e Add to create a new protocol using the Scan Protocol wizard
61. OWESS C Total Figure 3 2 Quantitate Window Run Easy Quant 2 Use the settings from the last session if you are using the same type of microarray or see Section 3 3 1 to change the settings If you make any changes be sure to adjust the template and register the images See Adjusting the Template and Registering Images on page 3 12 3 Click Start The ScanArray Express finds the spots a message box displays showing the progress of finding spots and then quantitates immediately 3 8 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Quant Chapter 3 3 3 1 Changing the Settings To change the Easy Quant settings refer to the table below for information Item Description Quantitation Type Template From GAL file From specifications From Arraylnformatics Quantitation Method Normalization Method Adjust Template and Register Images Select From Easy Quant This selection lets you enter or change settings or use the default or previous settings A spot template must be defined that matches the layout of the arrays on the selected scan Load a template from one of the methods listed below A template of circles appears superimposed on the microarray image you can then adjust and align the template Click this button to open the Open GAL File window where you can browse for and select a GAL file to use for your template For a description of GAL files
62. Sarwar de Oe eee See S 4 15 Runnings Line Stanie o cov saci seed eee seis Dost ates 4 19 Automatic Sensitivity Calibration 2 0 0 cece eee eee eee 4 20 Automatic Focus Calibration 2 gs02 cas 5 Gystindte ad Sentra erie Sala a SASS 4 22 Creating an Image Autosave Protocol 00 cece eee ene 4 24 Chapter 5 Quantitating with Protocols OVERVIOW ces i eae ee EA HEP RAR VS ae ee 5 1 Running a Quantitation Protocol 0 0 cece eee eens 5 1 Creating a Quantitation Protocol 4 5 65 426 pee euene pas Seed eee aaa as ees 5 3 The Quantitation Protocol Wizard 2 0 ccc e ee eee 5 4 Basic Information nnne tee eaten hg ee WAN GAD 4 Rn Oe A 5 5 mage Registration 0 4 44 stn aid Ba Pewee Edw ahd Oo 4 awn le ye owe eh Y 5 5 Template w0 eae eae he Rela ed aae Rae ee ea EAS 5 6 Defining a Template Using a GAL File l ce 5 8 Defining a Template From Specifications 0 000 0000 ce 5 9 Quantitation and Normalization Method 0 000000 e ee 5 10 Quantitation Methods 4 5 oyesi44 2nages say sewada 40 as Maas 5 12 Adaptive CNL san ooh Bit PUN e he a oh aOR Wha gM ey Rata 5 12 Fixed AECL ori 5 ine Nich hectic TAM ends 2 8 bene Ae Petes era a eee es aw 5 13 Adaptive Threshold oti ahs ore seas be lee cuce rsi etrn dre aie Sled cel are ee na ene 5 15 Histograms ss Salsa Ba ra a Gell AE E EE ries 5 16 Normalization Methods nss Geniegad He bees COGS ee eS 5 17 Creating a Spreadsheet Autosave Proto
63. Scan Next Fluor a Focus pasion 048 a z Laser power 80 PMT gain 4 aa Mouse is at 14395m 14155m Raw pixel intensity 0 Loaded microarray Fluorophore Cy3 1 of 2 Loaded Scan Next Fluor Focus position 0 s Controls for adjusting Laser power 80 Laser power and PMT gain PMT gain 80 4 Figure 4 13 Quick Scan Image 6 The scanned image displays on the workstation The name of the fluorophore is identified on the tab of each image You can change the focus position laser power and PMT gain during the scan using the up arrow and down arrows in the bottom right corner of the window 7 Click Scan Next Fluor to scan the next fluorophore User Manual pbtsupport perkinelmer com 4 17 Chapter 4 Scanning with Protocols To view the average intensity of a certain area click the Draw Circle from the Center Side button This enables you to draw a circle around the desired area using the mouse The average intensity of spots within the circle is displayed 8 ScanArray Express Composite Cy3 Control Cy5 Log Diagnostics Help 2 Zoomin Zoom Gut Zoom Full Scan Quantitate Ful Side Configure amp File Laver 4 488 nm Not Installed Laver 514 rm Not installed Autoloader Spot Viewer Composite oor Not Installed Load Status Not Installed Arraylnloimatios Not Installed Palette Source Black threshold
64. Step window select the second option A Scan Protocol and a Quantitation Protocol as shown in the following figure Protocol Group Step 1 This step executes A scan protocol Select a scan protocol and an image autosave protocol ion protocol Select a scan protocol animage and a spreadsheet autosave protocol Scan protocol Image autosave protocol Quantitation protocol Spreadsheet autosave protocol Name Name Name Name Dr Thomas Test1 Dynamic Repeatability Geo Slide 32x32 Demo Geometric Rotated Uniformity Static Repeatability 4 L E Bp t Ls Protocol description Protocol description Protocol description Protocol description li BDyesStd Test Quantitation Protocol Test Spreadsheet Protocol Protocols beginning with cannot be executed with current hardware CE j Figure 5 2 Creating a Step for Scanning and Quantitating 2 Select a Scan protocol Image autosave protocol Quantitation protocol and Spreadsheet autosave protocol 3 Click OK to save the step to the Protocol Group 4 Repeat for each step that you want to add to the protocol group You can add as many steps as you want 6 3 Running and Creating Batch Sets Batch Sets perform scans on one to twenty slides in an autoloader cassette If you haven t done so already you need to create a Batch Set definition that specifies which slots in the cassette hold microarrays and which protocol or protocol group to run on the m
65. Sypro Ruby Texas Red TO PRO 3 TO PRO 1 TOTO 3 TOTO 1 YO PRO 1 YOYO 1 and the Molecular Probes logo are trademarks of Molecular Probes Inc FluorX is a trademark of Amersham Biosciences Ltd Copyright 2002 PerkinElmer Life Science Inc All rights reserved No part of this manual may be reproduced or transmitted in any form without the written permission of PerkinElmer Printed in the U S A Part No 80M 826 Rev B Information in this document is subject to change without notice and does not represent a commitment on the part of PerkinElmer Contents Preface Contents of This Manual 0 0 ccc cece eee eee eens Vili Conventions Used in this Manual 0 00 00 00 ccc cc eee eee ees ix Where to Get Help 4 svat kdd ah iaiia We Re aak aa EEEE A BEE t ix For Additional Information 0 0 00000 cece eee eee ee eee ix Getting Help for Windows 2000 XP_ 0 cece ene nes x Chapter 1 Introduction OVET EW a ian ne ote 2 Os aA asa ee ee E Oe eo i as 1 1 The ScanArray Express System 0 0 0 0 cece cee eens 1 1 The Scanning Instrument sede ses ihe ied ioe 8 es de ROG KO 1 2 CICA ITN LEC ohn rs areas als be aes dd eh cost ite Oe dacs ob dines lk DR ek E eto din 1 2 The ScanArray Express os538 5 3 5 hg Se ah Re Ros ad ae RG aS 1 3 The ScanArray Express HT nica ea DVile ha Pela ne Daled Opinnd dheRea eae d 1 3 The ScanArray Express in an Integrated Microarray Laboratory 1 3 The User TCV I ACE is bo
66. To access the Tools 1 Open the Scan Protocol Tools window shown in Figure 4 11 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 Scan Protocol Tools Help and Getting Started The tools on this page can be used to adjust the focus position and also the laser powers and PMT gains of the individual fluorophores All of these tools perform specialized types of scans when they are run and require the presence of a microarray in the scanner Autoloader slot from which to load microarray 1 20 7 Quick Scan This tool performs a scan of a selected area using selected fluorphores allowing the user to adjust the focus position laser power and PMT gain while the scan is being performed Line Scan This tool repeatedly scans a single scan line to help determine the appropriate focus position and the appropriate laser power and PMT gains for the various fluorophores Automatic Sensitivity Calibration This tool scans selected areas of a microarray with selected fluorophores to determine appropriate settings for laser power and PMT gain Automatic Focus Calibration This tool scans a single scan line with a single fluorophore and determines the appropriate focus position Finish Cancel Figure 4 11 Scan Protocol Tools Window Each of the tools is described in a section below To use these tools the instrument must be connected and a microarray inserted
67. WORE ae 8 4 Appendix A Supported File Formats OVEIVIEW uaa aaa a Ae eas Raden pa Fs Bas oP eS as Ba A 1 Gene Array List gal Format ia sy etka tae BERS a eons Pas ao eee dae A 1 Description of File Format nannan nnana a aeneae A 2 Description of the Header SeCU OI ies pa ena Ry ig doa ei a eae ages A 2 Description of the Spot Data Section nannan cee A 3 Blocks S b rrays sk esre ceense ren oiie nan a e a a wa eb abi A 4 Example GAL file ete edhe elie rien a ae O ie RE A 4 GPR Results File Format ee snenc cei doania Wow eines vided Bhs Oa aa A 5 GPR Header son dneni dareen a i a een Be Soe eae eee A 5 GPR Dat ouea a tie glk a a E A E E a a a N A 6 CSV Results File Format act cox cas ek rno gh dee Coca A ae OP he xe eG ER A 9 Description of the File Format Description of the File Fields 2 44s assnegeenessn oes PIE Files n 6h Le Mah Rite a theca a T ate 5 ad erat ara au ava Appendix B Declaration of Conformity Electrical and Mechanical Safety Standards Laser Safety Standards y occ u43 tewearerecwiedce se has Electromagnetic Emissions Standards Electromagnetic Immunity Standards FCC Label for Class A Products 0 000000 e eee ICES 003 Label for Class A Products 04 Other EU Conformance lt 4 6 345 fine rove ad eae eae Ms Appendix C Specifications Appendix D Formulas ONEIVIEW att hi daa kee Hata
68. a B 7 Other EU Conformance The product is CE marked in conformance with the following directives 89 336 EEC A92 31 EEC A93 68 EED EMC Directive Europe 72 23 EEC A93 68 EEC Low Voltage Directive Europe B 2 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express User Manual Specifications Appendix The following specifications are for ScanArray Lite ScanArray Express and ScanArray HT The specifications are the same for each model except where noted Table C 1 Specifications ScanArray Specifications Description Sample Size Scanning Field Pixel Resolution Excitation Wavelengths Emission Wavelengths Sensitivity Scan Speed Dynamic Range Repeatability and Uniformity Image File Formats Palette Selections Workstation minimum specs Size Scanner External Laser pbtsupport perkinelmer com Standard or metric slides Width 0 97 1 02 24 6 25 5 mm Length 2 95 3 0 75 0 76 2 mm Thickness 0 037 0 051 0 93 1 29 mm 22 mm x 60 mm Norm 22 x 73 allowed User selectable for each acquisition 5 10 20 30 and 50 microns User selectable depending on the model Lite 543 and 633 nm Express and HT Up to 5 of 488 514 543 594 612 and 633 nm User selectable depending on the model Lite 570 and 670 nm Express and HT 508 522 530 549 570 578 592 614 660 670 and 694 nm Lite 0 05 fluorescent molecule ym Express and HT lt 0 1 fluore
69. a scan protocol display as shown in Figure 4 1 above The settings for performing the scan including the scan area are included in the scan protocol itself Unlike when using Easy Scan you cannot change the scan area or settings in this window You can set or change settings when running the scan as described in section 4 2 1 3 Select your scan protocol and image autosave protocol You can obtain this information from barcodes on the microarrays See Other Scan Settings on page 4 3 4 Click Start The scan protocol scans the microarray and saves the images using the Image autosave protocol The images are displayed in the Main Window 4 2 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 4 2 1 Other Scan Settings You cannot change the protocol settings from this window but you can change some of the settings for how the Scan is to run including getting the protocol information from barcodes To change the scan protocol settings refer to the table below for information Item Description Scan type The scan types that display depend on your system Without an autoloader the types are Run Easy Scan Run a scan protocol Run a protocol group On an HT autoloader system the types are Run Easy Scan and Run a batch set Scan area The scan area has been defined in the scan protocol If you want to Obtain scan protocol from barcode Scan Protocol Obtain image autos
70. able for information The default value of each item is the value specified in the protocol s parameters Item Description Scan resolution um The minimum and maximum resolutions are 5 and 50 microns Focus position um This is the distance from the lens to the calibrated nominal focus Fluorophores Check the fluorophores you wish to use for the scan PMT Gain This value can be changed at any time during the scan Enter the PMT Gain for each selected fluorophore Laser Power This value can be changed at any time during a scan Enter the Laser Power for each selected fluorophore Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 3 Set the area to be scanned by clicking Show Zoom Area and drawing a rectangle using the mouse on the image of the slide in the Quick Scan window Alternatively enter the X and Y coordinates if preferred Click OK to return to the Quick Scan window The scan area will default to that specified in the protocol s parameters 4 Click Start 5 The Quick Scan image displays ScanArray Run Composite Cy3 Contiol Cy5 Source Log Diagnostics Help aE Scan Stop Zoom In Zoom Out Zoom Full Dragging with mouse Draws Circle from Side Remove Circle Configure amp File Ele Configure Spot Viewer Composite Palette Black threshold 4000 Full color threshold j 583K SiS Median Restore
71. ady close to what you want Or click e Delete after selecting a protocol to delete a protocol 5 4 The Quantitation Protocol Wizard The Quantitation Protocol wizard opens with the Basic Information window displayed as shown below From this window using the wizard you can display and set all of the quantitation parameters You can move through the windows by clicking the numbers on the left or by using the buttons on the bottom of the window Quantitation Protocol Zoomed View Click the numbers to move from one window display to another 1 Basic Information 2 Template 3 Quantitation and Normalization i summay 1 Basic Information Protocol name Description EZ Quant default protocol 2 Zoom In Zoom Dut Zoom Full Figure 5 2 The Quantitation Protocol Wizard e Click Next to move to the next window and Back to move to the previous window e Click Finish to save all of your changes and close the wizard e Click Cancel to close the wizard without saving any of your changes You can change the image display making it larger or smaller using the Zoom buttons 5 4 Technical Support 800 551 2121 or 617 350 9263 ScanArray TM Express Quantitating with Protocols Chapter 5 Zoom In Zoom Out Zoom Full Click these buttons to zoom in or zoom out the display or to restore the display to the full slide view These controls remain visible and selectable throughout
72. all Columns Hide all Columns Show all Columns Hide all Columns in all Categories in all Categories in this Category in this Category Cancel l 2 Check the corresponding boxes for the columns you wish to show or hide To quickly select entire groups click on the appropriate Category 3 Click OK to implement the changes User Manual pbtsupport perkinelmer com 3 17 Chapter 3 Using Easy Scan and Easy Quant To set or change quality measurements 1 Click Set Quality Criteria to set or change quality measurement criteria The following window opens Set Quality Measurement CICH X Quality measurement method Signal to noise Signal to background Quality Measurement Parameters Maximum footprint um 100 Minimum signal to noise ratio 3 Lower limit Multiplier 400 1 70 Apply Set Criteria as New Default Eancel 2 Set the criteria you want to use The default method is Footprint Refer to the following table for information Item Description Quality Select one of the methods below Footprint is the default method measurement menog The formulas used by the different methods use the settings in the ScanArray Express Application Settings window The formulas are provided in Appendix D Footprint This is the distance between the expected position of a spot and its Signal to noise Signal to background Quality Measurement Parameters actual position Choosing this method
73. amples GeoSample32x32 Cy5 tif C Program Files PerkinElmer ScanArray Express Samples GeoSample32x32 Cy3 tif PMTVolts 600 600 NormalizationFactor RatioOfMedians 0 819944 NormalizationFactor RatioOfMeans 0 848769 NormalizationFactor MedianOfRatios 0 843367 NormalizationFactor MeanOfRatios 0 427747 NormalizationFactor RegressionRatio 1 0055 Jpegimage RatioFormulation W2 W11 543 633 pbtsupport perkinelmer com data fields columns Type of ATF file Date and time when the image was acquired The name of the settings file that was used for analysis The Array Content List file used to associate Names and IDs to each entry Type of scanner used to acquire the image User entered file comment Resolution of each pixel in um Name of each image buffer File name for each image buffer In a multi image tiff file the same name is repeated The voltage of the PMTs during acquisition The Ratio of Medians Normalization Factor The Ratio of Means Normalization Factor The Median of Ratios Normalization Factor The Mean of Ratios Normalization Factor The Regression Ratio Normalization Factor Name of optional compressed image saved with the results file The ratio formulation of the ratio image showing which image is numerator and which is denominator Page A 5 Appendix Entry Supported File Formats Description Barcode 00331 ImageOrigin 0 0 JpegOrigin 390 4320 Creator ScanA
74. anArray Specifications Descnpiion Other EU Conformance The product is CE marked in conformance with the following directives 89 336 EEC A92 31 EEC A93 68 EED EMC Directive Europe 72 23 EEC A93 68 EEC Low Voltage Directive Europe Specifications as of September 2002 PerkinElmer Life Sciences reserves the right to change these specifications without notice User Manual pbtsupport perkinelmer com Page C 3 Appendix C Specifications C 4 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Formulas D 1 Overview Appendix This chapter lists the formulas used by ScanArray Express for Quantitation D 1 1 Quality Measurement Formulas ScanArray Express calculates a set of raw measurements and marks spot quality based on the method and corresponding threshold values specified in the application settings see Chapter 7 and reports them in the export file Next to each quality measurement below are its raw measurement and criterian for marking spots as good Table D 1 Quality Measurement Formulas li riterian for markin Quality Raw Measurement Calculation t a orma g spots as Measurement Good Footprint For each subarray calculate the difference Spots with a calculated footprint less than the between the center of the nominal spots and maximum specified in the application settings the center of the found spot then shift the are good nominal spots by the difference Let the sh
75. anArray Express can read barcodes on the microarrays Using barcodes as unique identifiers on individual substrates provides a valuable way for tracking samples and workflow and automating analysis of microarray experiments Using barcodes allows a gene sample to be tracked through the entire microarray process including printing hybridization scanning quantitation and visualization For systems that are integrated with ArrayInformatics unique barcodes on microarrays are required and allow you to retrieve information from and automatically save data to the ArrayInformatics database 7 4 1 Supported Barcodes The ScanArray Express barcode reader supports Code 128 Code 39 and Interleaved 2 of 5 ITF symbologies by default The ScanArray Express barcode reader may be configured to support additional symbologies with help from technical support The maximum number of digits supported by each symbology depends on the minimum element width the barcode pitch in the printed barcode Alphabetic and numeric digits can be combined but alphabetic digits take more space than numbers thus decreasing the maximum number of allowed digits Table 7 1 lists the maximum number of alphanumeric characters for substrate barcodes Table 7 1 Substrate Barcodes Element Width Number of Characters Inches Mm Code 128 Code 39 ITF 010 254 mm 6 2 6 0075 19 mm 12 4 10 005 127 mm 22 8 16 User Manual pbtsupport perkinelmer com 7 9
76. ar So gota eed a hee ang WMP ORY ENE Reet Oa ER 1 3 The ScanArray Express Main Window 0 0 cece cence ences 1 4 Workflow for Scanning and Quantitation nosaus eee eee eee 1 5 The Different Ways to Scatti i4ae cies eee GG ae GA een oe ewe 1 6 Easy Scans ccc keenest oma wend ee tA Rae eee hee Ga See ee 1 6 Scan Protocols aaa e 9G 4g waste eae bg deed AE 4 Simoes tw N ea ea ANT 1 6 Protocol Groups and Batch Scans 0 cece eee eee 1 6 The Different Ways to Quantitate co 2c0 4vatiadasecenwaeaeohieiasda s 1 7 Easy CAIN ses cis ore aided aren bg hee gdh A hdl gy aoe Faded A Aah a Bae ae 1 7 Cuianititation PIOOcOhe 0 46 Aetauig dd edonne ede ew as Re OREO RES 1 7 Automatically Quantitating after Scanning 00 0000 0s 1 7 Preparitig to San 43 ses ell gs ene se eee a Rates edo eae Wes eee RS 1 8 Turning on the ScanArray Express and Warming the Lasers 1 8 Loading the Microarray or Cassette o n unnan nnana cee 1 9 Exiting the ScanArray Express Software 0 cece cece eee teens 1 9 Chapter 2 Getting Started Scan and Quantitate the Geometric Test Slide OVETVIEW Ci seh gal Lita eae hg etn ta aac charge E a e E eit nca cars Ast ath a Ghia 9X 2 1 Scanning the Geometric Test Slide 23 sia fywidwd aetteteeueetewdd aves 2 1 Wel Wit OLGA OES ns n n a2 a 6 chau a 4 209 Wd a aed Renee sae DS 2 3 improving the Image Display v4 0 gcse a4 oe o eGR A RAG SESS 2 5 Changing the Palette for t
77. atics GAL file from which to read the gene list and spot statuses Bbc0032 HomeXchange S amples GeoS ample3 Quantitation method Easy Quant Adaptive circle Adaptive threshold settings Fixed circle C Histogram Adaptive Circle Options Current options match defaults Normalization method LOWESS C Total Adjust Template and Register Images 3 Load an array content template in the form of a GAL file e click From GAL File e In the Open GAL File dialog box that opens browse to C Programs Files PerkinElmer ScanArray Express Samples and choose the file GeoSample32x32 gal The path may be different if the ScanArray Express software was installed in a folder other than the default click Open A template of circles appears superimposed on the microarray images Note When scanning the Geometric Test Slide you don t have to adjust the template When scanning your own slides you need to adjust the template as described in steps 4 and 5 For more information see Adjusting the Template and Registering Images in Chapter 3 User Manual 4 Click Adjust Template and Register Images This opens a Zoom window where you can align the template precisely and register the images if necessary pbtsupport perkinelmer com 2 7 Chapter 2 8 9 Getting Started Scan and Quantitate the Adjust the template by dragging it with the mouse until it is aligned with
78. ation method 5 16 Image autosave protocols creating 4 24 Image file formats C 1 Image poor uniformity 8 4 Images changing the palette 2 5 improving image display 2 5 registering 3 14 5 5 saving a portion 3 7 saving or printing 3 7 viewing 2 3 Instrument setting network options 7 16 shutting down 1 10 Instrument dimensions C 1 Instrument Laser excitation filter 1 2 Laser power setting 4 10 Index Laser safety C 2 Line scan running 4 19 LOWESS normalization 5 17 Mask for quantitation 5 12 Microarray jammed slide 8 3 Normalization method LOWESS 5 17 Total 5 17 Palette changing 2 5 Pixel resolution See Scan resolution PMT gain 4 10 Protocol groups creating 6 3 description 6 1 running 6 2 Protocols for quantitation 5 1 for scanning 4 1 image autosave 4 24 protocol group 4 1 5 1 spreadsheet autosave 5 19 Quantitation different ways to quantitate 1 7 saving results 4 1 5 1 Quantitation method adaptive circle 5 12 adaptive threshold 5 15 choosing 5 11 fixed circle 5 13 histogram 5 16 Quantitation Protocol 5 1 creating 5 3 defining atemplate 5 4 running 5 1 spreadsheet profile 4 1 5 1 wizard 5 3 Quantitation Protocol Wizard basic information 5 4 quantitation methods 5 10 summary 5 18 template 5 6 Quantitation Results CSVfile format A 9 GPR file format A 5 Quantitation results distribution plot 3 20 scatter plot 2 10 spreadsheet 2 9 viewing 2 9 Q
79. ave protocol from barcode Image autosave protocol Perform automatic quantitation Quantitation Protocol Spreadsheet Autosave Protocol Automatically Save in Arraylnformatics change the scan area you must select a different protocol Check this box to read the barcode of the microarray for the appropriate scan protocol If a barcode reader is not installed or if the barcodes are not configured this box is dimmed and unavailable to select See Configuring Barcodes in Chapter 7 To select a scan protocol click the Select a Scan Protocol button and select a protocol from the List of Scan Protocols window that opens The button on the Scan window fills with the name of the protocol Check this box to read the barcode of the microarray for the appropriate image autosave protocol If a barcode reader is not installed or if the barcodes are not configured this box is dimmed and unavailable to select See Configuring Barcodes in Chapter 7 To select an image autosave protocol click the Select an Image Autosave Protocol button and select a protocol from the List of Image Autosave Protocols window that opens The button on the Scan window fills with the name of the protocol Make sure this box is not checked unless you want to automatically quantitate after scanning See Automatic Quantitation after Scanning on page 4 4 Checking this box enables the following fields for selecting a quantitation protocol and spr
80. batch set to modify an existing set e Duplicate makes a duplicate of the highlighted batch set to help you create a new batch set from one that is already close to what you want Or click Delete after selecting a batch set to delete the set 6 8 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Automating Scans and Quantitation Chapter 6 4 Click Add The Batch Set window opens Batch Set Name LEST Description TEST Slot Status Protocol Group IV Read scan protocols from barcodes IV Read image autosave protocols from barcodes IV Send email upon completion of scan Email address Save in Arrayinformatics Image Spreadsheet Scan Protocol Autosave Protocol Quantitation Protocol Autosave Protocol 20 19 From Barcode From Barcode 18 a 16 15 14 From Barcode From Barcode Configure a Range of Slots items beginning with cannot be executed with current hardware Figure 5 3 Batch Set Window 5 Enter a name for the batch set and specify parameters for the set and configure each slot to be used Refer to the following table for information Item Description Name Enter a name for the batch set use a name that will help you identify the batch set Description Enter a description for the batch set Read scan protocols from barcodes Read image autosave protocols from barc
81. ce using it to ensure that undesirable pixels are not included in the results You should first run some quantitations and look at the View Spot Pixels in the Spreadsheet tab to understand what is being included in your quantitation results To use the histogram method 1 Select Histogram and click Set Histogram Options Quantitation Options Histogram Method Histogram for the Average Spot Patch Cy3 Frequency Plotted fluorophore Cys Signal Level Histogram Key The plot line shows the histogram for the selected fluorophore The background range is drawn as a black rectangle The signal range is drawn as a dark green rectangle You should select ranges that do not overlap If the ranges do overlap the overlap area is drawn as a dark red rectangle Percentile Ranges Signal range Background range 5 16 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Quantitating with Protocols Chapter 5 2 Set the signal range and background range Refer to the following table Item Description Signal range Low High Background range Low High Enter the percentile for the following values where percentile is the percentage of pixels in the sample Enter the minimum percentile value for the calculation of the spot statistics Enter the maximum percentile value for the calculation of the spot statistics Enter the minimum percentile value
82. code Units X Units Per Pixel Y Units Per PixeL X Offset Y Offset Status An example of the following rows ch1 F Program Files Packard BioChip DSOMA ExperimentSets qa 4 Images TAMRA TIF TAMRA04030270 EOO0CS Microns 20 00 20 00 0 0 Control Image END IMAGE INFO BEGIN NORMALIZATION INFO Normalization Method Normalization method Normalization Factor normalization Factor END NORMALIZATION INFO BEGIN DATA The first row contains the labels and the following rows are the measurements one for each spot Note Chx in the following columns represents Channels x x 1 2 3 4 The labels of the columns Following columns present for each spot Index Array Row Array Column Spot Row Spot Column Name ID X Y Diameter F Pixels B Pixels chx Footprint Flags Following columns Repeated for each channel Chx Median Chx Mean A 12 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Supported File Formats Chx SD Chx B Median Chx B Mean Chx B SD Chx gt B 1SD Chx gt B 2 SD Chx F Sat Chx Median B Chx Mean B Chx Signal Noise Ratio Following columns Repeated for Each Non Control channel Chx Ratio of Medians Chx Ratio of Means Chx Median of Ratios Chx Mean of Ratios Chx Ratios SD Chx Rgn Ratio Chx Rgn R Chx Log Ratio Following 2 columns are present if there 2 or more channels quantitated Sum of Medians Sum of Means Appendix Following Columns Repeated
83. col 0 c eee eee eee eee 5 18 Chapter 6 Automating Scans and Quantitation Overview ny hart fete eri VE ee ae al eat eda att A et amet bh ene ale Ce Ly 6 1 Running and Creating Protocol Groups 0 c eee eee eee 6 2 Creating a Protocol Group os 64 ae ja tmcee a annann eare 6 3 Running and Creatine Batch Sets aicas yc cue ei Rae Roe 8 hal Se IVE hala 6 6 Creating Batch Set 144 cceua iia e a gay aes Cau ae aes 6 8 Co feunng AS lOc ycz 5 2 they iria Sead aE OS ISS ES 6 10 Configuring a Range Of Slots a5 0r2e58 Sh Sah we oa SO am 6 11 Chapter 7 System Settings OVERVIEW ater iin r dad alot ea aa i Cae eae ee wae ea aaa aa 7 1 ScanArray Express Settiigs lt 5 aru unnan AG eee He ta eeeen on tangs 7 2 Connection Settings n susunan are gd ahs Ss ee eda ood 7 2 SCANNING Setting Ss an tees aka oe ai oid Wee wees Boas Sn dole een BS 7 2 Quantitation Settings 44 04 44 sande teeeu4e ada aeusle eed ae ckaeadgaeas 7 4 Arraylnformatics Settings oasis dette aie tA etree nneur 7 5 Other Settings rrgs hed a othe Bod a ued eee at tet Le ote a 7 6 Configuring Fluorophores 0 00 e een n eee 7 7 Defining a Fluorophore 4 2004 saosin de Oe eae Rea eae ae eens 7 7 Contivdring Barcodes 244 20ideaavadate poy eee ete COG deers 7 9 Supported Barcodes dics eaieds Svea w see a aa Rae ea dO Rs eG eS 7 9 Defining Barcode Parameters ys lt nnn dence acorns 5 a ane 8 Sane Pagers 7 11 Database Maintenance
84. ctions The ability to register images is provided so that you can test the quantitation template The amount by which you rotate or shift the images will NOT be saved as part of the quantitation protocol If the image you would like to shift is not part posite image click the button Set Source for Composite and select the image you wish to shift The first image in the composite the control image can be rotated but cannot be shifted Figure 5 3 The Quantitation Protocol Image Registration Window Click the Composite tab and adjust the Source experiment image to the Control To register the next image click Set Source for Composite Image and register it to the Control image in the Composite tab Do this for each image that needs to be registered 4 Click Next or click Template to go to the next window 5 4 3 Template Specify the template to be used by this protocol A template must be defined that matches the layout of the array on the scanned image You can import a template from a GAL file or you can manually define the array pattern If you use a GAL file it can be a local file or on a network drive or it can be accessed from ArrayInformatics for ArrayInformatics users The advantage of using a GAL file is that it also imports gene names for the spots from the file The defined template overlays onto the scanned image a template of circles that is closely aligned to the microar
85. dinates Set Scan Area to Full Microarray Show Zoom window Select Run Easy Scan The scan types that display depend on your system You may see Run Easy Scan Run a scan protocol Run a protocol group or on HT autoloader systems you will see Run Easy Scan and Run a batch set Select a scan resolution Scan resolution represents the individual pixel size in the scanned image and can be set to 50 30 20 10 or 5 microns Higher settings provide a quicker scan with reduced detail The default resolution is 10 microns For instruments with an autoloader specify the slot number of the microarray to be scanned The default is 1 If your system does not have an autoloader this field is dimmed and unavailable Select one first checkbox or two fluorophores to be scanned and set the PMT Gain for each Check the box for a fluorophore and click the button below it to open the Select a Fluorophore window Find and select your fluorophore in the list and click OK The window closes and the button fills with the fluorophore name Set a value for the PMT Gain for each selected fluorophore The value defaults to the value used in the last scan session NOTE Increasing the PMT Gain percentage improves sensitivity but also increases background noise and causes saturation A PMT Gain of approximately 50 percent lowers the background The laser power is set in the ScanArray Express application settings and cannot be changed here The
86. disk space to extend the amount of RAM available to a program Windows 2000 supports virtual memory and allows you to specify the amount of hard disk space to use as virtual memory Windows 2000 also manages the swapping of data from RAM and the hard disk space so that it appears to your application program s as though the virtual memory is RAM This reduces the amount of physical RAM required ScanArray Express uses RAM to save image data The following chart provides a guide for the amount of additional virtual memory to allocate on your workstation when running other applications concurrently with ScanArray Express Scans Additional Virtual Acquisition Scan Resolution Memory Required 1 5 microns 185 MB 1 10 microns 45 MB 2 5 microns 245 MB 2 10 microns 60 MB 3 5 microns 305 MB 3 10 microns 75MB 4 5 microns 365 MB 4 10 microns 90 MB 5 5 microns 425 MB 5 10 microns 105 MB To change Virtual Memory 1 Login as Administrator 2 Select Start Settings System 3 Select the Performance tab 4 Click the Virtual Memory Change button The three axes associated with the sample X is across the narrow dimension of the slide Y is the length of the slide Z is perpendicular to the slide In ScanArray Express lines are in the X direction Lines are selected by moving the slide in Y and focus is lens movement in Z User Manual PBTsupport perkinelmer com Index GAL files format A 1 GPR files format A 5 TIFF f
87. e Display Area Click a tab to view images or results Run Cyanine 3 Control Log Diagnostics Help Zoom In Zoom Out Zoom Full Dragging with mouse Draws Circle from Side Scan Quantitate Remove Circle Configure amp File Eile FullSide Configure Not Installed Spot Viewer Laser5 Cyanine 3 514 nm Not Installed Palette Black threshold 2320 Full color threshold 45K Loaded microarray 3x3 Median Unadjusted None Mouse is at 14305m 12915m Raw pixel intensity 2166 Click File to open or save a file Click Configure to create protocols protocol groups and batch sets and to configure system settings Figure 1 2 The ScanArray Main Window 1 4 Technical Support 800 551 2121 or 617 350 9263 ScanArray TMEX press Introduction Chapter 1 1 3 Workflow for Scanning and Quantitation The ScanArray Express performs two basic operations scanning and quantitation Depending on a workflow that you choose scanning and quantitating can be done separately and individually with manual or automatic saving of results or the two operations can be partly or completely automated using protocols and protocol groups A Batch Set is used for scanners with autoloaders The diagram below is an overview of the ways to scan and quantitate and points you to information about each Scan Click Scan and select from See Different Ways to Scan
88. e all of the images a Op Open Image Set from ESE T AATE Save Save in Save As Array Informatics Save All Save Portion of Image Close View Header Close All Print Set Control Image 2 In the Save Image dialog box that opens enter a filename and pathname for the image 2 7 Saving the Results You can save the results as a GPR file or as a CSV file The format of these files is described in Appendix A To save the results 1 Click the Spreadsheet Scatter Plot or Distribution Plot tab 2 On the Main Window click File in the Configure amp File box then click Save and provide a filename and file type 2 8 Scan and Quantitate Your Microarrays You have completed scanning and quantitating the Geometric Test Slide and saved the results Use the same process for your own microarrays specifying at each step along the process the settings that are appropriate for your microarray See Chapter 3 for more information on selecting settings for Easy Scan and Easy Quant Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Chapter 3 Quant Chapter Summary Overview 3 1 Using Easy Scan 3 1 Using Easy Quant 3 8 Viewing the Quantitation Results 3 14 3 1 Overview The Easy Scan and Easy Quant settings once set remain set until you change them This makes it easy to scan microarrays with the same spot configuration and quantitate the resulting images using the same settings
89. e barcode or label toward you slide the microarray into the slot until it touches the back The ScanArray Express will automatically align the microarray To load a cassette into the autoloader If you have a ScanArray Express system with an HT autoloader you can load up to 20 slides in the cassette Pull the cassette straight up and out Inside the cassette are fingers that hold the slide in place To prevent breaking these fingers load the microarray slides from the back of the cassette Insert a microarray face up into each slot you want to use slot 1 is on the bottom slot 20 on the top The barcode or label should be oriented so that it is toward the front of the instrument when the cassette is replaced in the autoloader Align the microarrays on the front of the cassette using the metal alignment block that ships with the autoloader The slides must be aligned to be recognized Replace the cassette into the autoloader and close the door 1 7 Exiting the ScanArray Express Software User Manual You can exit the ScanArray Express user interface and the system saves the current settings pbtsupport perkinelmer com 1 9 Chapter 1 Introduction To exit the ScanArray Express 1 Click the X in the top right hand corner of the ScanArray Express Main Window The following message displays Exiting ScanArray Express Exit the program 2 Click Yes to exit 3 You may leave the computer or instrume
90. e in the calibration 1 Allophycocyanin APC Fluorophore cannot be scanned with current hardware Settings Average spot size um 100 Target signal intensity 90 Sensitivity adjustment method C Keep laser power fixed vary PMT gain Keep PMT gain fixed vary laser power cost Figure 4 16 Automatic Sensitivity Calibration Window 2 Select the fluorophores to include in the calibration and specify the settings Refer to the following table Item Description Check the Check each fluorophore to be included and adjust the calibration area fluorophores to for each fluorophore if necessary include in the calibration Adjust the Calibration Select a fluorophore and click this button to adjust the area in the Area for the Selected window that opens Fluorophore Average spot size Enter a value The default is 100 Valid values are 75 um to 500 um um Target signal intensity Enter a value The default is 90 Valid values are 50 to 100 Sensitivity Select a method to use Adjustment Method Keep laser power fixed vary PMT gain This is the default Keep PMT Gain fixed vary laser power User Manual pbtsupport perkinelmer com 4 21 Chapter 4 Scanning with Protocols 3 Click Start to run Automatic Sensitivity Calibration It can be cancelled at any time by clicking Stop then Cancel 4 A progress bar displays 100 when complete Upon completion review the results and click
91. eadsheet autosave protocol You must select an already defined Quantitation Protocol Click the Select a Quantitation Protocol button to select a protocol from the List of Quantitation Protocols window that opens You must select an already defined Spreadsheet Autosave Protocol for saving the quantitation results Click the Select a Spreadsheet Autosave Protocol button to select a protocol from the List of Spreadsheet Autosave Protocols window that opens Check this box to automatically save the scanned image files to the specified location If your system is not integrated with Arraylnformatics this box is dimmed and unavailable to select User Manual pbtsupport perkinelmer com 4 3 Chapter 4 Scanning with Protocols 4 2 1 1 Automatic Quantitation after Scanning To automatically quantitate the images after scanning 1 Inthe Scan window select your scanning settings then check the Perform automatic quantitation box 2 Select a quantitation protocol and spreadsheet autosave protocol 3 Click Start The ScanArray Express scans the microarray and saves the images As the image is completed ScanArray Express immediately starts spot finding using the template called for in the quantitation protocol and starts quantitating When quantitation is finished ScanArray Express saves the results 4 3 Creating a Scan Protocol To create a scan protocol 1 Onthe Main Window click Configure in the Configure amp File group
92. ed data e Total uses the intensity of each spot in relation to all spots See Normalization Methods on page 5 17 4 Click Test Spot Finding to ensure that the template you have set up will find the spots 5 When you are finished click Summary to see a summary of the quantitation protocol 5 4 5 Quantitation Methods After a template is imported and adjusted in the protocol wizard Template window the ScanArray Express determines the center for each spot and determines the corresponding patch The patch is a rectangle that is constructed around the center of the spot with the dimensions indicated in the template Both the spot and the background must be defined with the patch The quantitation method selected then constructs masks for the spot and the background A mask is a pixel by pixel map that indicates the property of each pixel Background inner Spot mask _ Background outer Spot diameter Background Patch _ 5 4 5 1 Adaptive Circle The Adaptive Circle method fits all spots in the image with circles the circle diameter is estimated separately for each spot on the microarray with a minimum and maximum spot diameter as specified in the Adaptive Circle Options dialog box Adaptive Circle is the best method to use when all circles are not the same size This is the default method Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Quantitating with Protoco
93. efer to the table below and set the parameters Item Description Dimensions Maximum spot Enter the maximum spot diameter in microns diameter Inner background Enter the inner background diameter in microns This must be larger diameter than the maximum spot diameter Outer background Enter the outer background diameter in microns This must be larger diameter than the inner background diameter IMPORTANT The inner and outer background dimensions can never be equal pbtsupport perkinelmer com 5 15 Chapter 5 Item Quantitating with Protocols Description Spot Identification p Value Calculate Default Values The p value parameter controls the confidence for testing whether some spot pixels differ statistically from the eight median pixels in the background mask The p value represents a probability that the sampled eight spot pixels associated with an adaptive threshold are from a different population than that of the eight median background pixels The smaller the p value the more confident the derived spot threshold All pixels above the threshold are used for the spot intensity calculation Calculates the default values based on the array pattern selected in the microarray The defaults for your microarray depend on the template that is loaded 3 Click OK 5 4 5 4 Histogram Using the Histogram method the histogram for each spot is calculated Use caution with this method if you don t have experien
94. em Settings 7 2 ScanArray Express Settings You can dynamically change most software settings without restarting the instrument You must have local administrator privileges to change these settings To set or change application settings 1 Inthe Configure menu click Application Settings The Application Settings dialog displays Application Settings Other Array Informatics Connection Scanning Quantitation Connection to Instrument Specify IP address C Specify computer name Instrument IP address 10 Computer name 3 Cancel Figure 7 1 Application Settings Dialog Box 2 Click the tab for the settings you want to set or change and refer to the appropriate section below 3 Click OK when you are finished with all settings that you wish to set or change 7 2 1 Connection Settings On the Connection tab select either Specify IP address or Specify computer name The appropriate field becomes active where you can enter the IP address or the computer name The application must be restarted for the new instrument IP address computer name to take effect 7 2 2 Scanning Settings You can allow the user to skip the laser warm up and set default focus position and laser power for Easy Scan 7 2 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Settings Chapter 7 To set the scanning settings 1 Click the Scanning tab Application Settings Connection Settings
95. enables the Maximum footprint field below Uses the ratio of the spot intensity to the standard deviation of the local background of all spots on the microarray Choosing this method enables the Minimum signal to noise ratio field below Spots with a low signal to noise ratio can be quickly identified for later review Uses the comparison of the mean of spot intensity to the mean of local background Choosing this method enables the Lower Limit and Multiplier fields below The following fields will be enabled or dimmed and unavailable for selection depending on which method you select 3 18 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Quant User Manual Item Chapter 3 Description Maximum footprint um Minimum signal to noise ratio Lower limit Multiplier Enter the maximum size of the footprint in microns This measurement helps to distinguish spots from artifacts on the slide The default setting is 0 The minimum maximum settings are 100 through 72 000 This field is enabled when you choose the Signal to noise method The default value is 3 Valid values are 1 through 10 000 For Signal to background method Valid values are 0 through 64 000 More accurate spot intensities can be produced with a low intensity uniform background around all spots For Signal to background method The valid values are 1 through 65000 To set the status of a sp
96. equirements and Users Guide for Laser Safety IEC 825 Class I Laser Product B 3 Electromagnetic Emissions Standards FCC Part 15 Class A Radiated and Conducted USA EN55011 1991 Class A Radiated and Conducted Europe ICES 003 Industry Canada Interference Causing Equipment Standard Digital Apparatus Class A B 4 Electromagnetic Immunity Standards EN50082 1 Electromagnetic Compatibility Generic Immunity Standard Part 1 Residential Commercial and Light Industry EN61000 4 2 Electrostatic Discharge EN61000 4 3 Radiated Electromagnetic Fields EN61000 4 4 Electrical Fast Transient Burst User Manual PBTsupport packardbioscience com B 1 EN61000 4 5 Surge Immunity Requirements EN61000 4 6 Conducted Disturbances Induced By Radio Frequency Fields EN61000 4 11 Voltage Dips Short Interruptions and Voltage Variations Immunity Tests B 5 FCC Label for Class A Products This device complies with Part 15 of the FCC Rules Operation is subject to the following two conditions This device may not cause harmful interference This device must accept any interference received including interference that may cause undesired operation B 6 ICES 003 Label for Class A Products This Class A digital apparatus meets all requirements of the Canadian Interference Causing Regulations Cet appareil num rique de la classe A respecte toutes les exigences du R glement sur le mat riel brouiller du Canad
97. er in regard to products or parts furnished by third parties such being subject to the warranty of their respective manufacturers Service under this warranty shall be requested by contacting your nearest PerkinElmer Life Sciences office This warranty does not extend to any instruments or parts that have been subject to misuse neglect or accident or have been modified by anyone other than PerkinElmer Life Sciences or have been used in violation of PerkinElmer Life Sciences instructions The warranty is null and void if the cover has been removed from the system The foregoing obligations are in lieu of all other obligations and liabilities including negligence and all warranties of merchantability or fitness for a particular purpose or otherwise expressed or implied in fact or in law and state PerkinElmer Life Sciences entire and exclusive liability and buyer s exclusive remedy for any claims or damages in connection with the furnishing of goods or parts their design suitability for use installation or operation PerkinElmer Life Sciences will in no event be liable for any special incidental or consequential damages whatsoever and PerkinElmer Life Sciences liability under no circumstances will exceed the contract price for the goods for which liability is claimed This warranty shall be governed by and construed and enforced with the substantive laws of the Commonwealth of Massachusetts This warranty may vary outside of the United States In o
98. eriment 2 Double click a fluorophore to change it or click one of the following Add to add a new fluorophore Change after selecting a fluorophore to modify an existing fluorophore Duplicate makes a duplicate of the highlighted fluorophore to help you create a new fluorophore from one that is already close to what you want e Remove after selecting a fluorophore to remove it from the protocol pbtsupport perkinelmer com 4 9 Chapter 4 Scanning with Protocols 3 When you click Add the Fluorophore window opens Fluorophore Laser power PMT gain Fluorophore 5 FAM Alexa 488 Alexa 532 Alexa 546 Alexa 555 Alexa 568 Alexa 594 Alexa 647 Alexa 660 Allophycocyanin APC BODIPY 530 550 BODIPY 558 568 BODIPY 564 570 BODIPY 630 650 Calcein x gt Fluorophore cannot be scanned with current hardware Cancel Figure 4 7 Fluorophore Window 4 Select a fluorophore and specify the Laser Power and PMT gain e Increasing PMT gain improves sensitivity but also increases background noise and causes saturation The optimal signal to noise is obtained using 70 80 for the PMT Gain Leave the laser at 90 unless scanning under special conditions 5 Click OK to accept the changes and return to the protocol wizard 6 Click Finish if the PMT Gain and laser settings are known for this scan and will be kept constant for all scans performed with this Scan Protocol No further configuration
99. escription Show Hide Spot by Lets you select spots to hide or display depending on their status Status Show Hide Columns Let you select which columns of information to show or hide Set Quality Criteria You don t need to set this for the Geometric Test Slide For more information see Viewing the Quantitation Results in Chapter 3 Set Spot Status Lets you change the status for a spot Good Bad Found Not Found Absent View Spot Pixels Shows which pixels were treated as part of the spot during quantitation 2 5 2 Scatter Plot The Scatter Plot allows you to see any column of data plotted against any other column Composite Cy3 Control Cy5 Source Spreadsheet Scatter Plot Distribution Plot Log Help Run Scan J Cy5 Mean Spot vs Cy3 Mean Spot Quantitate A Configure amp File 5 Os Ls a TTTTT T A S LL rror oki Seta 469 at 10000 20000 30000 40000 50000 50000 rioan ray jene name Paks Shown Spots Properties Cy3 Mean Spot Zoomin Zoom Out Zoom Ful Unknown When you click a data point on the scatter plot the selected data point displays in the spot viewer and is highlighted in both the Spreadsheet and Image views when you switch back to those tabs The data is linked in all views 2 10 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Getting Started Scan and Quantitate the Geometric Test Slide 2 5
100. fferent area of the microarray may be used to adjust the sensitivity of each fluorophore The area to be used for a single fluorophore is shown as a darted rectangle To change the fluorophore select a different line from the list of fluorophores To change the outlines of the area use the same directions as were given on page 2 for changing the scan area Figure 4 8 Scan Protocol Sensitivity Calibration Areas Window User Manual pbtsupport perkinelmer com 4 11 Chapter 4 Scanning with Protocols To set the area to be used for automatically calibrating the signal intensity select a fluorophore and click Show Zoom Window The Zoom window allows greater accuracy when defining an area Setting as small an area as possible is recommended In the Zoom window draw a rectangle on the slide image using the mouse or enter the Area Co ordinates Sensitivity Calibration Area for Fluorophore FITC Zoom Window 5x Help amp Directions Composite Cy5 Conton Cy3 Source Symbols and Colors p The area of to be used for sensitivity calbration is shown as a darted rectangle Zoomin Zoom Out Zoom Full To Re Draw the Sensitivity Move the mouse to the upper left comer of the area you would ike to use Press the left most mouse buton down and hold it down Then move the mouse to the lower right comer of the area you would ike to use and release the mouse button To Modify the Sensitivity Calibrati
101. for less conventional applications such as tissue arrays and three dimensional substrates Item Description Focus position Do not change the focus position for conventional microarrays Start Automatic If you have selected a spot on the sample that has measurable Focus Calibration signals for the selected fluorophore in the protocol click Start Automatic Focus Calibration to automatically determine the optimum lens position A progress bar displays indicating automatic focus calibration Upon completion click Accept Changes to accept the adjustment or click Cancel to cancel the adjustment If the signal trace approaches 100 reduce the Laser Power and or PMT Gain or re run the automatic focus 5 Click Start review the results and click Accept Changes to save the results to the protocol 4 5 3 Automatic Sensitivity Calibration Only experienced users should use this tool The Automatic Sensitivity Calibration tool scans selected areas of a microarray with the selected fluorophores to determine appropriate settings for laser power and PMT gain 4 20 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 To perform Automatic Sensitivity Calibration 1 Click Run in the Automatic Sensitivity Calibration area of the Scan Protocol Tools window The Automatic Sensitivity Calibration dialog box displays Automatic Sensitivity Calibration ol x Check the fluorophores to includ
102. g only The available scan protocols and image autosave protocols display The following example shows the selection for scan protocols only the fields for quantitation are dimmed and unavailable for selection Protocol Group Step 1 This step executes A scan protocol Select a scan protocol and an image autosave protocol A scan protocol and a quantitation protocol Select a scan protocol an image autosave protocol a quantitation protocol and a spreadsheet autosave protocol Scan protocol Image autosave protocol Quantitation protocol Spreadsheet autosave protocol Name Jo Name Name ooo Dr Thomas Testi Dynamic Repeatability Geo Slide 32x32 Demo Geometric Rotated Uniformity Static Repeatability is ot Ls is Protocol description Protocol description Protocol description Protocol description 16DyesStd i Protocols beginning with cannot be executed with current hardware C ca j Figure 5 1 Creating a Step for Scanning 2 Under Scan Protocol select the protocol you want to include in this step of the protocol group 3 Under Image Autosave Protocol select an Image Autosave Protocol to use in this step 4 Click OK to send your selections to the Protocol Group window You can add as many steps as you want to the Protocol Group User Manual pbtsupport perkinelmer com 6 5 Chapter 6 Automating Scans and Quantitation To select scan and quantitation protocols 1 Inthe Protocol Group
103. ges Click Close to close the List of Image Autosave Protocols window 4 26 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Quantitating with Protocols Chapter 5 Chapter Summary Overview 5 1 Running a Quantitation Protocol 5 1 Creating a Quantitation Protocol 5 3 The Quantitation Protocol Wizard 5 4 Creating a Spreadsheet Autosave Protocol 5 18 5 1 Overview A Quantitation Protocol lets you set up parameters for quantitating including advanced quantitation methods and save them to be used again A Spreadsheet Autosave Protocol is used to save the quantitation results the protocol specifies the folder file naming convention and file type to use when saving the results Quantitation protocols can also be included in protocol groups or batch sets that are run from the Scan window See Chapter 6 Automating Scans and Quantitation for more information on protocol groups and batch sets To quantitate automatically after a scan see Running a Scan Protocol Automatic Quantitation in Chapter 4 This chapter provides instructions for running a Quantitation Protocol and for creating Quantitation Protocols and Spreadsheet Autosave Protocols iY Tip If you are a new or infrequent user you can use Easy Quant to become familiar with the quantitation process before creating and using Quantitation Protocols See Chapter 3 for information on using Easy Quant For a tutorial that leads you through
104. ght values in millimeters for X and Y The default scan area is the entire slide or 22 mm for width and 73 mm for height The default start X and Y positions are 0 00 mm The minimum width and height for the scan area are 0 1 mm and 0 05 mm respectively 3 Click Next to display the Scan Protocol Fluorophores window 4 8 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols 4 4 3 Fluorophores User Manual Chapter 4 In the Scan Protocol Fluorophores window you can select or change fluorophores to be included in the scan protocol and set a percentage for the PMT Gain and Laser Power You can also change the order of the listed fluorophores The first fluorophore listed is the control fluorophore To select Fluorophores 1 Open the Scan Protocol Fluorophores window Scan Protocol Fluorophores PMT Fluorophore Gain Laser Modify Power Add Change Duplicate Remove Move ToTop Up Down To Bottom Fluorophores beginning with cannot be scanned with current hardware NOTE By default the first fluorophore in the list will be used as the control for quantitation Figure 4 6 Scan Protocol Fluorophores Window Finish Cancel The window displays the current order of scanning and the name PMT Gain and Laser Power for each fluorophore in the list The first fluorophore scanned is the control image the second one is the source image exp
105. he Selected Image 0 0 5 2 5 Quantitating the Geometric Test Slide 0 0 eee 2 6 Viewing the Quantitation Results lt 4 4 0c asac Sexe sora an eo AS Gare als SGA 2 9 Spreadsheeta s timor ited eA a RN IG i ite ig gt Rh aac dea 2 9 Scatter LOD si Sintra a ea la tek Re get hk a ale Mole meee E Aircel tt De gee 2 10 DistibuUtion Plot pistera pronka ded pen chee 4 SOL Pee dees Pens 2 11 Saving the Imagis ssion ek Oar ead ae a ah wate is RAR hand Pre ile amp 2 12 Saving the Results 2 4 eater dat aa aout Re REA ee Ee eS 2 12 Scan and Quantitate Your Microarrays 0 0 cece een ee 2 12 Chapter 3 Using Easy Scan and Easy Quant OVETVIEW a Die Pa urease oria a e og gre while ee held sas nian Bg 4 aae a a a a dew 4 55 Want 3 1 Using Easy Scan acusiud evades keca de Raw hye uae aes bee aoe 3 1 Changing the SetinGS tt ageewius a ie hull aG ewan ad Bod aioe E EA 3 3 Adjusting the Display seauies cbt sitaes Senna Wren thE hen a kod gp Wearanes SBag gS 3 4 Changing the Control Image or Source Experiment Image 3 6 Saving or Printing Images ceo cav det we eee d CAV SA Ra awe 3 7 Using Easy Quant susmi wakes aces acd a poe Wh A alae aad ee DG See ahd 3 8 Changing the Sens 4345 Sei ddds peed ae db pad t ead pea eee dag eaad 3 9 Defining PO IE po Be done 5 die caste Slee tor ae g re nly Sods anit dices hey Uae cdl Woo a e aed eh G8 3 10 Adjusting the Template and Registering Images 0 00000005 3 1
106. he front of the display An enlarged detail display of the image as shown in Figure 2 1 is in the center of the window an image of the Full Slide is on the right side Image display in Main Window Zoom buttons ScanArray Express Run Composite Cy5 Control Cy Source Log Diagnostics Help Scan Dragging with mouse Draws Circle Quantitate Remove J ra Not Installed Configure amp File Full Slide Laver 4 488 nm N Full slide with outline of the detail shown in the large Antal display oor Not Installed Spot viewer Cassette Load Status Not Installed Palette Source Black threshold 4000 z Full color threshold F o 537K Loaded microarray 3x3 Median Restore Mouse is at 65991m 17037um Pigel intensity ratio 0 31 S Note Figure 2 1 Image Display User Manual pbtsupport perkinelmer com 2 3 Chapter 2 Getting Started Scan and Quantitate the On the left side of the window the spot viewer shows enlarged details of the spots as the mouse cursor moves over them You can change the palette and adjust the display to see the spots better Adjusting the display does not change the data for quantitation nor the data in the file only the appearance of the image on the screen If there are two or more images you can set the control image and if three or more images you can change the source experiment image To navigate the
107. he image the Y coordinate in um of the center of the feature indicator associated with the feature where 0 0 is the top left of the image the diameter in um of the feature indicator ScanArray Express Supported File Formats Column Title Appendix Description F633 Median F633 Mean F633 SD B633 Median B633 Mean B633 SD gt B633 1 SD gt B633 2 SD F633 Sat F543 Median F543 Mean F543543 SD B543 Median B543 Mean B543 SD gt B543 1 SD gt B543 2 SD F543 Sat User Manual median feature pixel intensity at wavelength 1 633 nm mean feature pixel intensity at wavelength 1 633 nm the standard deviation of the feature pixel intensity at wavelength 1 633 nm the median feature background intensity at wavelength 1 633 nm the mean feature background intensity at wavelength 1 633 nm the standard deviation of the feature background intensity at wavelength 1 633 nm the percentage of feature pixels with intensities more than one standard deviation above the background pixel intensity at wavelength 1 633 nm the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity at wavelength 1 633 nm the percentage of feature pixels at wavelength 1 that are saturated median feature pixel intensity at wavelength 2 543 nm mean feature pixel intensity at wavele
108. hnical support personnel Service Features Calibration Calibrate Attenuator Density Calibrate Flatness Configuration Control of Instrument Software View Instrument Restart the Instrument Configuration Software and OS Set Instrument Shut down the Instrument Network Options Software Save Calibration Values Restore Calibration Values Save Instrument Shut down the Instrument Configuration Software and OS Restore Instrument Configuration Figure 7 6 Service Features Window 7 6 1 Descriptions Service features are described in the following table Feature Description Calibration Calibrate Used by service personnel to calibrate critical instrument parameters Calibrate Attenuator Density Calibrate Flatness Save Calibration Values Restore Calibration Values this is password protected to prevent improper usage Used exclusively by service personnel to calibrate the instrument attentuator Improper usage may cause incorrect operation of the instrument Intended for use under the direction of service or technical support personnel this feature allows calibration of the X Y stage flatness Please consult with technical support before running this feature Creates a file containing all instrument related calibration values It is recommended to save this file one time in the event of an instrument hard drive failure although the data is stored in the factory when the instrument is ship
109. icroarray in that slot See Creating a Batch Set on page 6 8 6 6 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Automating Scans and Quantitation Chapter 6 To run a batch set 1 On the Main Window click Scan The Scan window opens where you can select Run a batch set You cannot changes settings from this window including the scan area To make any changes you need to select a different batch set or edit the batch set Scan Areas Not Drawn Scan type C Run Easy Scan I Use barcodes for all microarrays Batch set Select a Batch Set To change the scan areas select a different batch set Scan areas are not drawn for batch sets 2 Click Use barcodes for all microarrays to get the protocol information from the microarray barcodes OR Click Select a Batch set Select a batch set from the window that opens and click OK The window closes and returns you to the Scan window the button fills with the name of the batch set 3 Click Start The ScanArray Express loads the microarray in the first configured slot and runs the specified protocol group or protocols The microarray is scanned and the image s are saved to the location defined in the image autosave protocol If quantitation was included for a configured slot ScanArray Express begins spot finding then quantitates and saves the results to the location defined in the spreadsheet autosave protocol The images and results files a
110. icrons with respect to the origin which is the top left corner of the slide Required Line Block Column Row Name Last line of the header containing column titles for the spot data records ID A 2 1 2 Description of the Spot Data Section User Manual The spot data section contains lines that describe each spot in detail It includes the block column and row numbers for spots as well as descriptive name and identifier information There is one text line for each spot in the microarray containing a field for each of the descriptive columns Each line contains tab delimited fields in the order described in Table A 2 Table A 2 Spot Data Field Description Block Block number for the spot Column Column where the spot is located within a block Row Row where the spot is located within a block Name Gene name limited to 40 characters ID Gene identifier limited to 40 characters pbtsupport perkinelmer com Page A 3 Supported File Formats A 2 2 Blocks Subarrays A block is a set of spots printed by one pin within a 4 5 mm x 4 5 mm square or a 9 mm x 9 mm square area Blocks are also called subarrays Blocks are numbered from top left to bottom right For example Spot 1 3 Spot 1 4 OO Spot 2 3 Spot 2 4 QO Spot 3 3 Spot 3 4 Spot 4 3 A 2 3 Example GAL file The following simple array list file describes four blocks BlockCount 4 each with 24 columns and 5 rows For simplicit
111. ie EEE A RORE eae i Quality Measurement Formulas Glossary Index Preface Contents of This Manual User Manual Preface Summary Contents of This Manual viii Conventions Used in this Manual ix Where to Get Help ix For Additional Information ix Getting Help for Windows 2000 XP_ x The following table describes the contents of this User Manual Chapter Description Chapter 1 Provides an overview of the ScanArray instrument hardware and software and an overview of the ways to scan and quantitate using ScanArray Express Chapter 2 Helps you become familiar with ScanArray Express by running an Easy Scan and Easy Quantitation on the Geometric Test Slide Chapter 3 Provides instructions for scanning and quantitating using Easy Scan and Easy Quant Chapter 4 Provides instructions for scanning using a protocol includes instructions for creating a Scan Protocol Chapter 5 Provides instructions for quantitating using a protocol includes instructions for creating a Quantitation Protocol Chapter 6 Provides instructions for creating and using Protocol Groups and Batch Sets to analyze multiple experiments and to automate your scanning and quantitating Chapter 7 Provides instructions to set system wide settings Chapter 8 Provides diagnostic and troubleshooting information Appendix A Formats of files supported by ScanArray Express Appendix B Specifications Appendix C Declaration of Conformity Appendix D
112. iew 6 1 Running and Creating Protocol Groups 6 2 Running and Creating Batch Sets 6 6 6 1 Overview Automating ties together all of the steps for scanning and analyzing your microarrays Using Protocol Groups or Batch Sets you can set up the ScanArray Express to automatically scan quantitate and save your results without intervention A Note This is in addition to some of the automatic file saving and automatic quantitation you can implement when running a single Scan Protocol as described in Chapter 4 User Manual Protocol Groups is a powerful new ScanArray Express feature A protocol group lets you run a series of scan protocols on a microarray one after the other without intervention and automatically save the results to a specified location In addition you can include quantitation protocols in the protocol group allowing you to scan then quantitate and save all scanned images and quantitation results to a specified location again without user intervention Scanning From a batch set lets you run a scan protocol different protocols or a protocol group on up to 20 slides in a cassette To use a batch set requires a ScanArray Express system with the HT autoloader option This chapter provides instructions for running a Protocol Group or creating a new one and for running a scan from a batch set or creating a new Batch Set pbtsupport perkinelmer com 6 1 Chapter 6 Automating Scans and Quantitation 6 2 Run
113. ifted nominal position be X Y the found position to be x y the footprint is X x Y yy Signal to Noise Spot intensity standard deviation of Spots with a signal to noise ratio greater than background intensity Signal to background Multiplier M Lower Limit L r4 M x background mean r2 L background mean the minimum specified in the application settings are good Spots with a mean greater than r4 anda mean greater than rz are marked good User Manual pbtsupport perkinelmer com Appendix D Formulas D 2 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Warranty PerkinElmer Life Sciences warrants to the customer that the ScanArray Express Microarray Analysis System will be free from defects in material and workmanship and will meet all performance specifications for a period of one year from the date of shipment This warranty covers all parts and labor In the event that the instrument must be returned to the factory for repair under warranty the instrument must be packed in the original packaging The outside of the package must indicate the Returned Material Authorization Number RMA provided by PerkinElmer Life Sciences PerkinElmer Life Sciences shall not be liable for any incidental special or consequential damage loss or expense directly or indirectly arising from the use of the ScanArray Express Microarray Analysis System PerkinElmer Life Sciences makes no warranty whatsoev
114. ignal range is drawn as a dark green rectangle You should select ranges that do not overlap If the ranges do overlap the overlap area is drawn as a dark red rectangle Histogram Key Percentile Ranges Signal range Background range Diameters Inner background diameter pm 1st Outer background diameter um 282 Calculate Default Values Cancel 2 Refer to the following table and specify your settings Item Description Plotted fluorophore Percentile Ranges Signal Low Signal High Background Low Background High Inner Background Diameter um and Outer Background Diameter um Calculate Default Values The highlighted fluorophore will be plotted Select the fluorophore Enter the minimum and maximum percentile values for calculations where percentile is the percentage of pixels in the sample The default is 5 valid values are from 0 to100 The default is 95 valid values are from 0 to 100 The default is 5 valid values are from 0 to 100 The default is 95 valid values are from 0 to 100 Inner and outer background diameter depends on the array pattern Calculates the default values based on the array pattern of the microarray The defaults depend on the template that has been loaded 3 Click OK Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Quantitating with Protocols Chapter 5 User Manual 5 4 5 3 Adaptive Threshold
115. iles description A 14 unable to open 8 4 Adaptive circle quantitation 5 12 Adaptive threshold quantitation 5 15 Additional information where to find ix ArrayInformatics description 1 3 Automatic focus calibration 4 22 Automatic sensitivity calibration 4 20 Automating automatic quantitation after scanning 4 4 using batch sets 6 1 using protocol groups 6 1 Backing up the database 7 13 Barcodes configuring 7 9 labels 7 10 supported symbologies 7 9 Batch sets creating 6 8 description 6 1 running 6 7 Calibrating automatic focus 4 22 automatic sensitivity 4 20 Contents of manual viii Control image changing 3 6 defined 3 6 Database backing up 7 13 Declaration of conformity B 1 Diagnostic tools 8 1 Diagnostics power on 8 1 Distribution Plot 3 20 dynamic range specification C 1 electrical safety C 2 electrical specifications C 2 electromagnetic emissions C 2 electromagnetic immunity C 2 Emission peak See Fluorophore 7 8 emission wavelengths C 1 EU conformance C 3 Excitation peak See Fluorophore 7 8 Experiment See Source image External laser troubleshooting 8 3 File formats CSV results A 9 GAL files A 1 GPR files A 5 GPR Results A 5 Fixed circle quantitation 5 13 Fluorophore configuring 7 7 defining anew 7 8 emission peak 7 8 Index excitation peak 7 8 trademarks 1 1 Geometric Test Slide quantitating 2 6 scanning 2 1 tutorial 2 1 Glossary 1 1 Histogram quantit
116. ion pm Start Automatic Focus Calibration a Start position Y mm 35 00 Line length mm 22 00 PMT Laser Fluorophore Gain Power Show Zoom window Cyanine 5 75 100 Clear Previous Plot Lines Stop Accept Changes Cancel Figure 4 15 Line Scan Window 2 Set the scan area and describe where the line is Item Description Start position X Y Enter the coordinates of the area to be scanned Line length Enter the length of the scan line Show Zoom Window Click to open the Zoom window where you can select an area with greater precision Using the mouse draw a line over the area of interest on the slide image The line should be through the middle of the selected dots User Manual pbtsupport perkinelmer com 4 19 Chapter 4 Scanning with Protocols 3 Select the fluorophore Change the PMT Gain and Laser Power while the scanning is in progress to find the optimal settings Item Description Fluorophore Select the desired Fluorophore PMT Gain Laser Change the instrument sensitivity by adjusting the Laser Power Power and PMT Gain settings so that the signal is within 90 of maximum to prevent saturation The Line Scan changes dynamically with each adjustment Clear Previous Plot Click this to clear the display Lines 4 Focus position rarely needs adjustment when using conventional genomic and proteomic microarrays printed on flat substrates Focus position may need to be adjusted
117. ion method Adaptive circle Adaptive threshold Set Histogram Options Test Spot Finding Normalization method LOWESS Total Help and Directions All of your changes to the quantitation method and the method options will be saved as part of the quantitation protocol Figure 5 5 Quantitation Protocol Quantitation and Normalization Window 2 Select a quantitation method Adaptive circle the preferred method uses a minimum and maximum spot diameter percentages of the nominal spot diameter to construct a mask See Adaptive Circle on page 5 12 Fixed circle uses the parameters of the spot diameter and background inner and outer dimensions to create a spot mask and background mask See Fixed Circle on page 5 13 Adaptive threshold uses the parameters of the spot diameter and background inner and outer dimensions to create a spot mask and background mask then refines the mask on a pixel by pixel basis See Adaptive Threshold on page 5 15 Histogram uses the same mask for both the spot and background and creates a graph of each pixel quantity versus the pixel intensity See Histogram on page 5 16 3 Select a normalization method LOWESS or Total User Manual pbtsupport perkinelmer com 5 11 Chapter 5 Quantitating with Protocols e LOWESS Locally Weighted Scatter Plot Smoothing carries out robust locally weighted scatter plot smoothing for both equally spaced and non equally spac
118. is required for the protocol Click Next if the maximum fluorescence values are significantly different from microarray to microarray While it will significantly increase individual scan time ScanArray Express can automatically calibrate each microarray for maximum fluorescence values with minimal saturation before scanning takes place 44 4 Sensitivity Calibration Areas In the Scan Protocol Sensitivity Calibration Areas window you can select an area of the microarray to be used to adjust the sensitivity of each fluorophore In this window you select the area in the next window Automatic Sensitivity Calibration you specify the settings for the calibration 4 10 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 The larger the area that you specify as the calibration area the more time it will take to perform the calibration To set the Sensitivity Calibration Area 1 Open the Scan Protocol Sensitivity Calibration Areas window Scan Protocol Sensitivity Calibration Areas Basic Information 3 Fluoropho Calibration y Calibration 6 Tools Sensitivity Area for Selected Fluorophore Show Zoom Window Area Area Width x Height ram Fluorophore Top Left ram 0 00 0 00 22 00 22 00 x 73 00 Fluorophores beginning with cannot be scanned with current hardware Copy Current Area to all Fluorophores Help amp Directions di
119. j Zl Ful color threshold HJ jsx Loaded 3x3 Median Auto Adjust Mouse is at 16823pm 21602um Pivel intensity ratio 1 91 Figure 4 14 Viewing Average Intensity of an Area 9 When the Quick Scan is done a Quick Scan Results window appears Quick Scan Results As part of Quick Scan you made the following changes Parameter New Focus Position um Cy3 Laser Power Cy3 PMT Gain Accept these changes as part of the scan protocol 10 Click Yes to accept the changes as part of the scan protocol Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Scanning with Protocols Chapter 4 11 To cancel a Quick Scan at any time during the scan click Stop on the Main Window 4 5 2 Running a Line Scan Line Scan should be used only by advanced users The Line Scan repeatedly scans a single scan line to help determine the laser power and PMT gains for the various fluorophores and displays the signal intensity for the specified line scan area on an oscilloscope like screen You can adjust the focus position Y start position laser power and or PMT gain while reviewing real time changes in signal intensity To perform a Line Scan 1 Click Run in the Line Scan area of the Scan Protocol Tools window The Line Scan dialog box displays Line Scan Signal vs X Position 9 10 11 12 13 14 15 16 17 108 19 0 X Position mm Start position X mm Focus posit
120. kstation running the ScanArray Express software User Manual pbtsupport perkinelmer com 1 1 Chapter 1 Introduction Figure 1 1 shows the ScanArray instrument models from left to right the ScanArray Lite the ScanArray Express and the ScanArray Express HT with 20 slide autoloader ep Snares The ScanArray Lite The ScanArray Express The ScanArray Express HT 1 2 Figure 1 1 The ScanArray Express Instrument Models All three models run the ScanArray Express software on the workstation The software is used to control all aspects of scanning quantitating and saving the scanned images and quantitation results using a network connection between the workstation and the instrument 1 1 1 1 The Scanning Instrument The instrument includes one to four lasers depending on the system configuration Usually lasers are mounted inside the scanner however large lasers or lasers with a significant power dissipation may be externally mounted The laser includes appropriate beam shaping optics an excitation filter a shutter and beam alignment optics Lasers turn off automatically after 120 minutes of inactivity to maximize the service life of the laser Optional add ons to the instrument include a barcode reader and autoloader 1 1 1 2 ScanArray Lite The ScanArray Lite has two internal lasers and can be upgraded with an optional barcode reader This model supports two Laser Excitation Wavelengths of 543 nm and 6
121. l does not turn on contact PerkinElmer 8 3 1 3 Microarray is Jammed If a microarray becomes jammed the ScanArray Express attempts to correct the problem with a PURGE sequence To run a manual PURGE sequence 1 The HT system includes sensors that monitor the location of the microarray If a microarray becomes jammed a PURGE sequence is initiated by the software that locates the jam and places the microarray back into the cassette An error message displays on the client computer 2 Ifa PURGE Sequence does not fully eject the microarray into the cassette a message displays and you will have to remove the microarray manually by removing the front cover removing the cassette and extracting the microarray from the system User Manual pbtsupport perkinelmer com 8 3 Chapter 8 Diagnostics and Troubleshooting 8 3 1 4 Poor Image Uniformity Contact Technical Support at 800 551 2121 or 617 350 9263 8 3 2 Software 8 3 2 1 Unable to Open Stored TIFF files 1 Make sure the stored file names do not include dots other than the dot preceding the file extension e g tif csv gpr slashes back slashes or dashes in the file name 2 Make sure there is enough free space on the hard disk to open the file If there is not locate any unnecessary image files and delete them to free up space 3 Ifyou are still unable to open the files contact PerkinElmer 8 4 Recording the ScanArray Express System Activity A s
122. levator Homing Moves the hardware to accurately locate the home sensor and applies the appropriate calibration Includes all initialization and homing of Y axis hardware Moves the hardware to accurately locate the home sensor and applies the appropriate calibration offset Downloads operational code to the GIM board Includes all initialization and homing of X axis hardware Moves the hardware to accurately locate the home sensor and applies the appropriate calibration Downloads operational code to the PAM board Verifies presence of 1 5 laser modules and determines type of laser internal external manufacturer Verifies serial communication exists with serial laser if one is installed Serial lasers include internal and external and are located in laser positions 4 and 5 A failure to communicate with internal serial laser 4 indicates a potential hardware problem Includes all initialization of the optional barcode reader This includes establishing serial communication with the barcode reader and downloading of all configuration parameters Verifies serial communication exists with the barcode reader Includes all initialization of the optional the HT autoloader This includes programming downloading configuration parameters and homing of the HT Downloads operational code to the HT controller board Downloads configuration calibration parameters to the controller Moves the elevator hardware to accurately loca
123. ls Chapter 5 User Manual To use the adaptive circle method 1 Select Adaptive circle and click Adaptive Circle Options Adaptive Circle Options Diameters Minimum spot diameter of nominal Maximum spot diameter of nominal Calculate Default Values J Cancel 2 Enter the minimum and maximum spot diameters These are not absolute values but are a percentage of the nominal spot diameter as specified in the GAL file or template OR Click Calculate Default Values to calculate the default values based on the array pattern selected in the microarray The defaults for your microarray depend on the template that is loaded 3 Click OK 5 4 5 2 Fixed Circle The Fixed Circle method fits all spots in the image with circles of fixed diameter All spots must be of the same size and shape Using the Fixed Circle Method the spot mask and the background mask are constructed using the parameters of the spot diameter and the background inner and outer dimensions pbtsupport perkinelmer com 5 13 Chapter 5 Quantitating with Protocols To use the fixed circle method 1 Select Fixed Circle and click Set Fixed Circle Options Quantitation Options Fixed Circle Method Histogram for the Average Spot Patch Cyanine 5 Frequency Signal Level Plotted fluorophore The plot line shows the histogram for the selected fluorophore Cyanine 3 The background range is drawn as a black rectangle The s
124. manufacturer of the fluorophore Once these values are entered the ScanArray Express automatically selects the laser and an appropriate filter for the fluorophore 7 To override the system selection click Advanced Options and enter the desired Laser and Filter then click OK Advanced Fluorophore Options Help and Directions These controls allow you to select which laser and filter will be used with this fluorophore overriding the choice of laser and filter which would normally be made by the software It is not recommended that you use this approach unless you are certain that you understand the consequences J Specify a filter Filter 4 570nm J Note Changing the filter or laser settings is not recommended as the ScanArray Express has optimized these selections based on the Excitation Peak and Emission Peak entered 7 8 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Settings Chapter 7 8 Click the Palette button to display the palette list Select the desired palette and click OK providing a spread of intensities instead of limited shades of just one color This allows A Note Using the rainbow palette for the simple images provides more visual data by you to differentiate more spots than you can with a single color 9 Click OK to close the Fluorophore dialog box and save the new fluorophore 7 4 Configuring Barcodes With an optional barcode reader installed the Sc
125. med Protocol group Scan protocol Image autosave protocol Quantitation protocol Spreadsheet autosave protocol Name Name Name Dr Thomas TEST Dynamic Repeatability i Geo Slide 32x32 Demo Geometric Geometric New 919 Rotated Uniformity i Static Repeatability ois His gt is Protocol group description Protocol description Protocol description Protocol description Protocol description 16DyesStd i tems beginning with cannot be executed with current hardware Cancel Figure 5 4 Cassette Slot Window The corresponding boxes are enabled for a protocol group or protocols and display a list of available protocols 3 Ifyou want to save your results automatically in ArrayInformatics check the box to save the images and results if quantitation is performed in ArrayInformatics If your ScanArray Express is not integrated with ArrayInformatics this box will be dimmed and unavailable to select 4 Select the protocol group or protocols to execute and click OK 6 10 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Automating Scans and Quantitation Chapter 6 6 3 3 Configuring a Range of Slots To configure a range of slots 1 Click Configure a Range of Slots on the Batch Set window The Range of Cassette Slots window opens Range of Cassette Slots First slot 1 20 IV Set slot status I Set use of Arraylnformatics J Remove the quantitation f 5 rotocol and spreadsheet Last slo
126. n Zoom Out Zoom Ful Template Properties Subarray Properties Spot Properties Dragging with mouse Moves or resizes all subarrays Rotates all subarrays C Moves a subarray C Moves a spot A Zoom to Subarray Register Images Image to shift or rotate ior Offset pile 0 0 qv J gt br Rotate Image 180 Set Source for Composite Image e Figure 3 4 Adjust Template and Register Images Window with Zoomed Display 2 Click on a subarray of the template The example above shows just one subarray The selected subarray turns blue and the Adjust Template group is activated You can move the highlighted subarray template by clicking Move a subarray under the Dragging with Mouse options then dragging the subarray along the substrate Or you can click Subarray Properties to enter specific information about the location of the subarray template and spots in the subarray Subarray Template Properties Subarray top left corner x um Subarray top left corner y pm J Straighten spot rows and columns in this subarray Cancel Figure 3 5 Subarray Template Properties Dialog Box pbtsupport perkinelmer com Chapter 3 Using Easy Scan and Easy Quant 3 To move a spot click Move a Spot under Dragging with Mouse Four blue triangles appear around the spot you can drag the spot in any direction to align it perfectly to the circle of the template 4 To
127. nEimer i Run Composite Cy5 Contra Cy3 Source Spreadsheet Scatter Plot Distibution Plot Log Diagnostics Help Scan General Cy5 Control Raw Spot Cy5 Control Mise ga FEE Subanay S Spot Spot S AER Quantitate Inder Center usesres Goon Na Cora Nare nese Kara Mace es peel pee re ee ee Std Dev Bkgnd Bkgnd eolrow colon eumy um i Bkgnd Bkgnd PKs g 1Std 2Std Laser 2 Configure amp File 2 LT 18 14458 56 i 3 WW He 200816 2 1445261 4 WW Hs 1257 1869 13897 1496348 Crime W S WW He 121876 2069 13837 Good 40045 46083 33499 546 S63 1438290 18551 1000 100C js WW Hs 41714 2269 13837 Good 40223 47598 39631 592 608 1441559 15582 1000 10C Laser 4 7 ii Hs 99873 2469 13837 Good 3910 46528 3883 573 605 13881 11 14225 1000 10C eee ES 8 Hs 79241 2679 13837 Good 45M0 48071 44813 597 593 1021338 14809 1000 100i ineasat Spot Viewer 3 v1 Hs 56023 2873 13837 Good 47266 50227 468650 6GIG 625 1044243 18850 1000 100i ro ie 10 v1 Hs 73853 3069 13837 Good 42223 48646 41646 577 598 1553303 15170 1000 100i Ei S Hs1104 3279 13837 Good 49123 50353 48528 60 BIS 8007 25 16011 1000 100 Installed 12 Ww Hs 6101 3473 13837 Good 49828 50235 43230 598 6I6 760820 16448 1000 100i 13 v1 Hs 39948 3679 13837 Good 49703 50478 43089 614 622 7071 20 17348 1000 100i 4 il Hs 87223 3873 13837 Good 49340 5054 48727 613 619 680031 15367 1000 100C 15 11 Hs73105 4079 13837 Good A964 49811 48579 585 598 579756 15403 1000 10
128. ngth 2 543 nm the standard deviation of the feature intensity at wavelength 2 543 nm the median feature background intensity at wavelength 2 543 nm the mean feature background intensity at wavelength 2 543 nm at wavelength 2 543 nm at wavelength 2 543 nm the percentage of feature pixels with intensities more than two standard deviations above the background pixel intensity at wavelength 2 543 nm the percentage of feature pixels at wavelength 2 that are saturated pbtsupport perkinelmer com Page A 7 Appendix Column Title Supported File Formats Description Ratio of Medians Ratio of Means Median of Ratios Mean of Ratios Ratios SD Rgn Ratio Rgn R F Pixels B Pixels Sum of Medians Sum of Means Log Ratio Flags Normalize F1 Median B1 F2 Median B2 F1 Mean B1 F2 Mean B2 SNR 1 F1 Total Intensity Index User Defined the ratio of the median intensities of each feature for each wavelength with the median background subtracted the ratio of the arithmetic mean intensities of each feature for each wavelength with the median background subtracted the median of pixel by pixel ratios of pixel intensities with the median background subtracted intensities with the median background subtracted the standard deviation of pixel intensity ratios the regression ratio the coefficient of determination for the current regression value the
129. ning and Creating Protocol Groups Once a protocol group is set up it is easy to select and run To create a Protocol Group see Section 6 2 1 To run a protocol group 1 Onthe Main Window click Scan The Scan window opens where you can select Run a protocol group Scan Areals Scan type Run Easy Scan Runa scan protocol Protocol group Select a Protocol Group IT Automatically save in Anaylnformatics To change the scan area s select a different protocol group 2 Click Select a protocol group and select a group from the List of Protocol Groups window that opens and click OK The window closes and returns you to the Scan window the button fills with the protocol group name 3 Click Start The ScanArray Express loads the microarray and runs the first protocol in the Protocol Group The microarray is scanned and the image s are saved to the location defined in the image autosave protocol If quantitation was included in a step of the Protocol Group ScanArray Express begins spot finding then quantitates and saves the results to the location defined in the spreadsheet autosave protocol The images and results files are closed and ScanArray Express runs the next protocol step 6 2 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Automating Scans and Quantitation Chapter 6 This process continues until all protocols in the group have run The images and results from the last prot
130. nt 3 3 1 1 Defining a Template Specify a template to be used for quantitation You can import a GAL file which also assigns gene names to each spot on the microarray or you can manually define the array pattern by entering values to describe the subarray and spots For a detailed description of a GAL file see Appendix A To define a template using a GAL file 1 In the Quantitation window under Template click From GAL File The Open GAL File dialog box opens Open GAL File Look in samples a Contents gal Files of type GAL gal Cancel 2 Highlight the desired file and click Open The name and location of the selected GAL file displays in the Quantitate window under the From GAL File button 3 10 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Quant Chapter 3 To define a template from specifications 1 In the Quantitation window under Template click From Specifications The Template Specifications window opens Template Specifications Subarrays Number of rows of subarrays Number of columns of subarrays Rotation degrees Horizontal pin spacing mm 45 90 Custom 250 Vertical pin spacing mm 45 390 C Custom 450 I Straighten rows and columns of subarrays Spots Rows of spots per subarray Columns of spots per subarray Horizontal spot spacing center to center ym l Vertical spot spacing center to center
131. nt running after exiting the ScanArray Express Note If you need to shut down the instrument use the Service Features as described below To shut down the instrument 1 Inthe Main Window click Configure in the Configure amp File group The Configure menu displays 2 Click Service Features then click Shut down the instrument software and OS 3 Wait for the Ready LED on the front of the instrument to turn off then wait 30 seconds and turn off the power switch on the right side of the instrument To shut down the computer 1 Your computer should automatically turn off If not click the Start menu on the task bar then click Shutdown A message displays on the monitor screen that it is okay to turn off the computer and you can turn off the power Technical Support 800 551 2121 or 617 350 9263 ScanArray TMEX press Getting Started Scan and Chapter 2 Quantitate the Geometric Test Slide 2 1 Overview Chapter Summary Overview 2 1 Scanning the Geometric Test Slide 2 1 Viewing the Images 2 3 Quantitating the Geometric Test Slide 2 6 Viewing the Quantitation Results 2 9 Scan and Quantitate Your Microarrays 2 12 This Getting Started chapter leads you through a typical scanning and quantitation session using Easy Scan Easy Quant and the Geometric Test Slide that ships with the ScanArray Express system The Geometric Test Slide is a test sample made of photo resist printed on glass 2 2 Scanning the Geome
132. o an electrical signal The signal is amplified filtered and converted to a digital value for each pixel The ratio of light energy to current for the PMT output The higher the Gain the smaller the amount of light needed for a given amount of current PBTsupport perkinelmer com Term Definition Protocol Regedit Repeatability Resolution Saturation Scanning protocol Spot Subnet Mask Substrate TCP IP Technical Support 800 551 2121 or 617 350 9263 A formula that defines an experiment or portion thereof See Scanning protocol QuantArray In Microsoft Windows 95 Windows 98 Windows NT and Windows 2000 and Windows XP operating systems the Registry is a single place for keeping information such as what hardware is attached what system options have been selected how computer memory is set up and what application programs are to be present when the operating system is started Regedit is an editing program used to update installed and uninstalled application programs It is also used to update the Registry file The change in light level detected at a given point on the slide between two scans Mechanical electronic and optical changes all affect repeatability The pixel size that is being scanned for one data point ScanArray Express can scan as small as 5 microns A fluorescence signal that is strong enough to drive the PMT detector to its full scale maximum voltage and output signal is said to cause
133. ocol are saved as well but remain displayed on the screen You can close these files or open any of the other images results that have been saved 6 2 1 Creating a Protocol Group To create a Protocol Group 1 From the Main Window select Configure in the Configure amp File group then click Protocol Groups The List of Protocol Groups window opens List of Protocol Groups Description Duplicate Delete View Usage Sort By Name Description Protocol group cannot be executed with current hardware 2 Sort the list optional by Name or Description 3 Click one of the following to open the Protocol Groups window Add to create a new protocol group Change after selecting a protocol group to modify an existing group If you change and save a protocol group that is currently in use the ScanArray Express uses the updated protocol group the next time that protocol group is called The protocol group currently being run continues with the original settings Duplicate makes a duplicate of the highlighted protocol group to help you create a new group from one that is already close to what you want Or click Delete after selecting a protocol group to delete the group View Usage to see where the protocol group is being used User Manual pbtsupport perkinelmer com 6 3 Chapter 6 To create a protocol group Automating Scans and Quantitation 1 Click Add The Protocol Group window opens A p
134. odes Send email upon completion of scan Slot Status Configure a Range of Slots Check this box to read information in the microarray barcode on which protocols to run Check this box to read the image autosave protocol information from the microarray barcodes Check this box to send an email after scanning has completed running the batch set enter an appropriate email address Set or change the status of each slot by clicking the Status button next to the slot number The options are Full Empty or Ignored The default is Empty See Configuring a Slot on page 6 10 Click this button to open the Range of Cassette Slots window where you can set the same parameters for an entire range of slots See Configuring a Range of Slots on page 6 11 User Manual pbtsupport perkinelmer com 6 9 Chapter 6 Automating Scans and Quantitation 6 3 2 Configuring a Slot To configure a single slot 1 Click the status button for the slot and change the status to Full The Cassette Slot Number window opens 2 Select the button for what you want to execute for this slot e a scan protocol e both a scan protocol and quantitation protocol or a protocol group a scan protocol Cassette Slot 20 This slot executes A scan protocol Select a scan protocol and an image autosave protocol protocol group Select only a protocol group I Save images in Arraylnformatics also save the spreadsheet if quantitation is perfor
135. on 1 0 Second line of an ATF file 8 number of optional header lines plus 1 5 number of spot data columns Type of file the same in all GAL files Optional Lines BlockCount 4 BlockType 0 URL Supplier PerkinElmer Life Sciences ArrayerSoftwareName SpotArray ArrayerSoftwareVersion 2 0 Blockn A 2 Technical Support 800 551 2121 or 617 350 9263 Number of blocks described in the file 4 in this example Type of block described rectangular in this example 0 rectangular 1 orange packing 1 2 orange packing 2 The URL for the Go To Web command The manufacturer that supplied the array or arrayer The name of the arrayer software The version number of the arrayer software The position and dimensions of each block There is one record for each block and each record contains 7 fields Each field is separated by a comma followed by a space X position of center of top leftmost spot in the block in um 2 Y position of center of top leftmost spot in the block in um 3 Nominal diameter of spots within the block in um 4 Number of columns of spots in the block ScanArray Express Supported File Formats Appendix Record Description 5 Column spacing of spots center to center in the block in um 6 Number of rows of spots in the block 7 Row spacing of spots center to center in the block in um Note Positions on arrays are measured in m
136. on Area Move the mouse over the edge that you would ike to ch The mouse pointer will change shape when you are close enough Press the leftmost mouse button down and hold it down release the mouse button Area Co ordinates Start position X mm 258 Stat position Ymm 13 38 Area with fom 358 Area height mt 300 Full Microarray Zoom to Area Figure 4 9 Drawing a Rectangle on the Slide Image Zoom Window The default scan area is the full substrate for each fluorophore A different area of the microarray may be used to adjust the sensitivity of each fluorophore or the same area may be used for all fluorophores in the protocol by clicking Copy Current Area to all Fluorophores Note Be sure to set the area for all fluorophores If you select an area for one fluorophore but neglect to set the area for all fluorophores any fluorophores not set will scan the whole slide the default which could take a long time possibly hours for dense arrays Technical Support 800 551 2121 or 617 350 9263 Click Next to display the Scan Protocol Automatic Sensitivity Calibration window ScanArray TM Express Scanning with Protocols Chapter 4 4 4 5 Automatic Sensitivity Calibration In the Scan Protocol Automatic Sensitivity Calibration window you can adjust the scanner so that the brightest features in a defined area of an array will have a chosen signal level The area was defined in
137. ot 1 Highlight the individual spot and click Set Spot Status The options are Found Not Found Absent Good or Bad Found Not Found Absent Good Bad 2 Select the status you want to assign To view spot pixels 1 Click View Spot Pixels xs indicate pixels which were treated as patt of the spot during quantitation The image shows which pixels were treated as part of the spot during quantitation pbtsupport perkinelmer com 3 19 Chapter 3 Using Easy Scan and Easy Quant 3 4 2 Viewing the Scatter Plot The Scatter Plot tab displays a scatter plot of spot brightnesses from both fluorophores Any data that appears on the spreadsheet can be selected and viewed in the scatter plot The axis measurements and scales can be changed by clicking on the axis label button Run Composite Cy3 Control Cy5 Source Spreadsheet Scatter Plot Distribution Plot Log Help so Cy5 Mean Spot vo Cy3 Mean Spot pan Quantitate F A f iscam J Laser2 Configure amp Fil Eie Configure Spot Viewer Cy5 Mean Spot I eryr retir rA errire 20000 30000 40000 50000 60000 Spot Inde 469 a ie name PAKS Shown Spots Properties Cy3 Mean Spot gt Zoom in Zoom Out Zoom Full Unknown Figure 3 7 Scatter Plot Tab Data can be filtered and displayed based on status by clicking the Shown Spots button at the bottom of the plot Set axis boundaries by clicking
138. p gt Perkin life sciences TM ScanArray Express Microarray Analysis System User Manual User Notice Users are advised that the use of this ScanArray instrument or the SpotArray instrument for certain applications in analyzing microarrays may require a license from one or more third parties that have patents in this area The following are some of the companies that have patents related to microarrays Affymetrix Inc Incyte Genomics Oxford Gene Technology Ltd Hyseq Inc There may be other third parties that have patent rights in this area PerkinElmer is not encouraging or suggesting that the users of this equipment employ this equipment in a way that would infringe on the patent rights of any third parties PerkinElmer suggests that users consult their own legal advisors for counsel on non infringing or licensed uses of this machine in analyzing microarrays The ScanArray Express is a registered trademark of PerkinElmer Life Sciences Inc Windows Windows 2000 and Windows XP are registered trademarks of Microsoft Corporation GenePix is a registered trademark of Axon Instruments Inc Alexa 488 Alexa 532 Alexa 546 Alexa 555 Alexa 568 Alexa 594 Alexa 647 Alexa 660 BODIPY 530 550 BODIPY 558 568 BODIPY 564 570 BODIPY 630 650 BODIPY TMR Calcium Crimson Calcium Green 1 Calctum Orange Magnesium Green Magnesium Orange Oregon Green 448 Oregon Green 500 Rhodamine Green Rhodamine Red Ribo Green SYBR Green
139. pbtsupport perkinelmer com 7 11 Chapter 7 System Settings Item Description Scan protocol ID is Select First lower barcode or Second upper barcode to indicate printed on whether the scan protocol ID information is on the lower or upper barcode on the slide Enter the starting position on the barcode and the length 1 through 13 of the scan protocol ID Barcodes cannot overlap and there must be no common characters 3 Click OK to save the barcode configuration 7 5 Database Maintenance The ScanArray Express database is where your protocols autosave protocols and data are stored Make a copy of the ScanArray Express database regularly to ensure that in the event the database becomes corrupted a recent backup copy of the database is available to restore Database maintenance is also used by the database administrator to backup restore and repair the database Database Maintenance Eq Restore Database Compact and Repair Database Last backed up Unknown Last compacted Unknown Figure 7 5 Database Maintenance Window Button Description Backup Database Allows you to backup the database Restore Database Allows you to restore the database Compact and Repair Allows you to compact and repair the database This feature the Database defragments the database in a way that is similar to defragmenting your hard drive for better space utilization and lets you use the smallest amount of space possible for
140. ped Used exclusively by service personnel Restoring invalid calibration values can cause hardware damage to the instrument 7 14 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Settings User Manual Feature Chapter 7 Description Configuration View Instrument Configuration Set Instrument Network Options Save Instrument Configuration Restore Instrument Configuration Control of Instrument Software Restart the Instrument Software and OS Shut down the Instrument Software Shut down the Instrument Software and OS Information pertaining to the instrument hardware what is installed version numbers and software version numbers See Figure 7 7 Allows the user to remotely change the network settings on the instrument See Setting the Instrument Network Options on page 7 16 Creates a file containing all instrument related configuration information It is recommended to save this file one time in the event of an instrument hard drive failure although all data is stored in the factory when the instrument is shipped See Figure 7 7 below Used exclusively by service personnel Restoring invalid instrument configuration information could cause improper operation of the instrument This is used to cause the instrument re initialize itself This should be used when the instrument is determined to be in an inoperable state and the user would typically cycle the inst
141. pots to plot from any one of the images or any images with the control image Select a Column to Plot Category Column General Cy5 Control Cy3 Ratio of Median Spot Ratio of Mean Spot Median of Pixel Ratios Mean of Pixel Ratios Regression of Pixel Ratios M Log2 Ratio of Median Spot M Log2 Ratio of Mean Spot A Mean of Log2 Median Spot 4 Mean of Log2 Mean Spot Ratio of Median Norm Spot Ratio of Mean Norm Spot Median of Norm Pixel Ratios Mean of Norm Pixel Ratios Regression of Norm Pixel Ratios M Log2 Ratio of Median Norm Spot M Log2 Ratio of Mean Norm Spot 4 amp Mean of Log2 Median Norm Spot A Mean of Log2 Mean Norm Spot Std Dev of Pixel Ratios p for Regression Ratios pbtsupport perkinelmer com 3 21 Chapter 3 Using Easy Scan and Easy Quant 3 4 4 Viewing Information About the Images You can view information about one of the displayed non composite images including whether or not it has been saved the filename if saved and the settings that were used To view image information 1 Click the tab for which you want to view information 2 On the Main Window click File in the Configure amp File group Open Image Set from __Aay Informatics Save in Array Informatics Save All Save Portion of Image Close View Header Close All i Print Set Control Image 3 On the File menu click View Header A Header Information window opens the example below is for the cu
142. press A GPR file supports only two fluorophores and does not save all of the data columns See the description below CSV A text file of quantitation results A csv file saves all information that is in the quantitation spreadsheet results TIF the images can be saved as TIF files as well as bitmap BMP JPEG and RAW files A 2 Gene Array List gal Format The gene array list format is written to an ASCII text file that is viewable by applications such as Microsoft Notepad and Wordpad The file contains header information and information for each spot in the microarray GAL files conform to the ATF format a standard tab delimited text file format readable by many downstream scanning and quantitation software applications They can be created in Microsoft Excel by saving an Excel spreadsheet as Text Tab delimited User Manual pbtsupport perkinelmer com A 1 Appendix A 2 1 Supported File Formats Description of File Format Gene array list files contain two major sections the header section and the spot data section A 2 1 1 Description of the Header Section The header section describes basic file information and provides information about each of the blocks Each line is explained in Table A 1 Record Table A 1 GAL File Format Description Required Lines ATF 1 0 85 Type GenePix Array List v1 0 First line of an ATF file the same in all GAL files File format ATF and versi
143. profile IDs Image profile ID is printed on i First Lov Second Upper barcode Location of the image profile ID within the first lower barcode Stat at MI 2 3 4 5 6 7 8 9 10 11 12 13 CT Length 1 2 35 6 7 8 9 10 11 12 13 IV Barcodes contain scan protocol IDs Scan protocol ID is printed on First Lower barcode Second Upper barcode Location of the scan protocol ID within the second upper barcode Start at RY 15 16 17 18 19 20 21 22 23 24 25 26 Length Figure 7 4 Barcode Dialog Box 2 Specify the settings referring to the following table for information Item Description Barcodes contain Check this box to indicate that the barcode includes the identification of image autosave the image autosave protocol to be used when saving the scanned protocol IDs image files Checking this box enables the barcode fields below it Image autosave Select First lower barcode or Second upper barcode to indicate protocol is printed on whether the image autosave protocol information in on the lower or upper barcode on the slide Enter the starting position on the barcode and the length 1 through 13 of the image autosave protocol information Barcodes contain Check this box to indicate that the barcode includes the identification of scan protocol scan protocol to be used when scanning this microarray Checking this information box enables the barcode fields below it User Manual
144. protocol Instrument Instrument Software IP Address Label Laser Microarray NIC Network Interface Card Palette Photo Bleaching Pixel PMI PMT PMT Photomultiplier Tube Board PMT Gain User Manual All of the information associated with an image including all system settings at the time a scan was started date and time the scan was performed instrument SN and revision instrument software revision client software revision and user comment field The information used to determine how images are stored This includes format and path The hardware that comprises the entire Scanner including the scanner module and the external laser This is the internal instrument software necessary to operate the instrument Network address of a workstation or network IP Addresses are always in the form of 4 number fields separated by dots Each number field represents 1 byte Values can be in the range of 0 255 The numbers on the left of the string define the network the numbers on the right define the individual workstation or Network Interface Card NIC A thin flexible adhesive backed piece of plastic or paper which a barcode or other identifying information may be recorded A device that amplifies light ScanArray Express has 2 5 lasers at different wavelengths which excite specific fluorophores A substrate with an array of spots printed on it the end product of a spotting instrument The microarray
145. ption Enter a description optional Pattern for File Check the box for each item you want to include in the file names The Names information you select will be included in the filename For example in the figure above the boxes are checked for Date and Time The fields are included in order selected resulting in a filename such as 080702_0819 GPR Custom Text Check this box to enable the edit field and enter custom text such as descriptive information to be included in the filename You can enter up to 64 characters File name pattern Shows the fields selected their order and the resulting pattern Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Quantitating with Protocols Chapter 5 Item Description File Types Select either one or both file types to save the data GPR saves the quantitation results as a Gene Results file CSV saves the quantitation results as an Excel spreadsheet Path to output files Enter the desired path for output files or use the Browse button to locate the path which can be a local path or a path on the network 7 Click OK to save the spreadsheet autosave protocol 8 Click OK to close the List of Spreadsheet Autosave Protocols window User Manual pbtsupport perkinelmer com 5 21 Chapter 5 5 22 Technical Support 800 551 2121 or 617 350 9263 Quantitating with Protocols ScanArray TM Express Automating Scans and Chapter Quantitation Chapter Summary Overv
146. r Histogram Method Low Signal Value Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Supported File Formats Appendix High Signal Value Low Background value High Background value For Fixed Circle Low Signal Value High Signal Value Low Background value High Background value Nominal Spot Diameter value Inner Background Diameter value Outer Background Diameter value For Adaptive Threshold Method Maximum Spot Diameter value Inner Background Diameter value Outer Background Diameter value p value value END QUANTITATION PARAMETERS BEGIN QUALITY MEASUREMENT CRITERIA Selected Criteria FootPrint SignalToNoiseRatio SignalToBackground MaximumFootprint value or MinSignalToNoise value Or LowerLimit value Multiplier value END QUALITY MEASUREMENT CRITERIA BEGIN ARRAY PATTERN INFO Units Microns Array Rows Array Columns Spot Rows 32 Spot Columns 32 Array Row Spacing 2000 00 Array Column Spacing 2000 00 Spot Row Spacing 201 04 Spot Column Spacing 200 52 Spot Diameter 100 00 Interstitial 0 0 is off 1 is first one missing 2 is second one missing Spots Per Array 1024 Total Spots 1024 END ARRAY PATTERN INFO User Manual pbtsupport perkinelmer com Page A Il1 Appendix Supported File Formats BEGIN IMAGE INFO The first row contains the labels and the following rows are the information one for each image Lables ImageID Channel Image Fluorophor Bar
147. ral scan protocols on a microarray one after the other without intervention and automatically save the results to a specified location In addition you can include quantitation protocols in the protocol group allowing you to scan then quantitate and save all scanned images and quantitation results to a specified location again without user intervention For more information see Chapter 6 1 6 Technical Support 800 551 2121 or 617 350 9263 ScanArray TM Express Introduction Chapter 1 Using a Batch Set lets you run different scan protocols or protocol groups on up to 20 slides in a cassette To use a Batch Set requires a ScanArray Express system with the HT autoloader option For more information see Chapter 6 1 5 The Different Ways to Quantitate Before starting review the ways you can quantitate your microarrays 1 5 1 Easy Quant The Easy Quant settings are persistent that is they remain from the last quantitation session So it s easier to quantitate the same type of microarrays For the new or occasional user Easy Quant is easy to use all user selectable settings are available from one screen and you can change your settings as you work Use Easy Quant if Your lab uses only one type of two color microarray e You want to walk up and use the equipment you can change settings as you work e You want to test your slide Easy Quant is a quick and easy way to do it 1 5 2 Quantitation Protocol
148. ray spots Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Quantitating with Protocols Chapter 5 To define a template 1 Open the Template window Quantitation Protocol x Composite Cy3 Control Cy5 Zoom In Zoom Out Zoom Full 3 Template Define a template p reTma From From GAL File Specifications _ Informatics From Anay GAL file from which to read the gene list and spot statuses Modify Template Subaray Properties Modify the ecient Moves or resizes all subarray Templ ate Finaida Moves a subanay Zoom to Subaray Help and Directions All of your changes to the template willbe saved as part of the quantitation protocol EXCEPT for adjustments you make to the locations of individual spots Figure 5 4 The Quantitation Protocol Template Window 2 Specify the source of the template Refer to the following table for a description of the possible choices Instructions for using a GAL file or specifications are in the sections following the table For a description of a GAL file see Appendix A After loading a template you may need to modify it in Step 3 Item Description Template Click a button to specify the source of a template From GAL file Click this button to open the Open GAL File window where you can browse for and select a GAL file to use Below the button the GAL file from which to read the gene list and spo
149. re closed and ScanArray Express unloads the microarray and proceeds to the next configured slot User Manual pbtsupport perkinelmer com 6 7 Chapter 6 Automating Scans and Quantitation This process continues until all microarrays in the configured slots in the batch set have been processed The images and results from the microarray in the last processed slot are saved as well but remain displayed on the screen You can close these files or open any of the other images results that have been saved After running a batch set you can check the log to verify that all microarrays were processed 6 3 1 Creating a Batch Set To create a batch set 1 On the Main Window click Configure in the Configure amp File group of the main window then click Batch Set The List of Batch Sets window displays List of Batch Sets Description Modify Dr Thomas Dr Thomas Add Dynamic Repeat Dynamic Repeatability Change Geo Slide 32x32 Geo Slide 32x32 Demo EA Geometric Geometric Rotated Uniformity Rotated Uniformity Static Repeatabil Static Repeatability Duplicate Description Batch sets beginning with contain scan protocols that cannot be executed with current hardware If these batch sets are run the incompatible protocols will be skipped 2 Sort the batch sets optional by Name or Description 3 Click one of the following to open the Batch Set window e Add to create a new batch set Change after selecting a
150. readsheet Autosave Protocols window Add to create a new protocol Change after selecting a protocol to modify an existing protocol Duplicate makes a duplicate of the highlighted protocol to help you create a new protocol from one that is already close to what you want Delete after selecting a protocol to delete it e View Usage after selecting a protocol to display a list of protocol groups and batch sets which include the selected protocol 4 Click Add User Manual pbtsupport perkinelmer com 5 19 Chapter 5 5 20 5 Quantitating with Protocols The Spreadsheet Autosave Protocol dialog box opens To create a unique filename you must include the date and time if a barcode is available it should be included in the filename Spreadsheet Autosave Protocol Description Pattern for File Names Information to include in the file names l Barcode JV Time hours minutes IV Date month day year J Custom text c File name pattern lt Date gt lt Time gt File types IV GPR Gene Results I CSV Excel spreadsheet Path to output files C Program Files PerkinE Imer S can4rray Express Ima Browse cancel Figure 5 7 Spreadsheet Autosave Protocol Dialog Box 6 Fill in the information as listed in the following table Item Description Name Enter a name for the spreadsheet autosave protocol Use your own name or some other text that makes the protocol easy to find Descri
151. register the images use the buttons and arrows in the Register Images box to move the selected image in the direction of the arrow you click until the images are aligned Register Images Image to shift or rotate Selected Image Cy5 Offset pixels 0 1 gt Rotate Image 180 Set Source for Composite Image 5 When the template is aligned and the images are registered click OK This returns you to the Quantitate window page 3 8 where you can make any additional changes or start your quantitation 3 4 Viewing the Quantitation Results Quantitation results display in the Main Window They include Spreadsheet tab with information about each spot and subarray on the scanned microarray including each gene name the accession number subarray location spot location and spot intensity values listed by fluorophore and normalized spot intensity values Scatter Plot tab lets you view spot intensity two channels at a time e Distribution Plot lets you see geographic trends for your microarrays areas of the slide that are being under or overwashed for example 3 14 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Quant Chapter 3 The views in all three tabs are linked a data point selected in one tab is automatically selected when you chi 3 4 1 Viewing the Spreadsheet ange to another tab An example of the spreadsheet is shown below
152. rotocol group is organized into a series of steps each step may include protocols for scanning or protocols for both scanning and quantitation Protocol Group Description Scans and quantitations to perform Each line is a Step Scan Protocol Image Profile in the Protocol Group Protocol cannot be executed with current hardware Quantitation Protocol Spreadsheet Profile Modify Add Change Duplicate Remove 2 Specify a Name and Description for the Protocol Group Use a name that will help you identify the group later In this window you can Add Change Duplicate or Remove a Step from the group A step is a selection of protocols to run each line in this window is a step 3 Click Add to add a Step The Protocol Group Step 1 window opens See To select scan protocols for a group on page 6 5 See To select scan and quantitation protocols on page 6 6 4 Once you have added all of the steps you want to include in the group you can re order them if you wish using the buttons in the Move group 5 When all protocols have been selected and they are in the order of steps in which you want the steps to run click OK to save the protocol group 6 4 Technical Support 800 551 2121 or 617 350 9263 ScanArray TM Express Automating Scans and Quantitation Chapter 6 To select scan protocols for a group 1 Inthe Protocol Group Step window select A scan protocol to create a step for scannin
153. rray Horizontal spot spacing center to center um Vertical spot spacing center to center um Spot diameter um I Straighten rows and columns of spots in all subarrays Completely Reset Modify Template CRA Template Using Changes Only 2 Provide the information for the array pattern as described in the following table Item Description Subarrays Describes the geometry of the microarray Number of rows and Enter the number of subarray rows and the number of subarray number of columnsof columns subarrays Rotation If the microarray is skewed on the substrate you can rotate the template to align the template to the microarray User Manual pbtsupport perkinelmer com 5 9 Chapter 5 Quantitating with Protocols Item Description Pin spacing Enter the horizontal and vertical pin spacing that was used to create the horizontal microarray Pin spacing vertical Straighten rows and columns of subarrays Spots Rows of spots and Columns of spots per subarray Horizontal spot spacing center to center um Spot diameter Straighten rows and columns of spots in all subarrays 4 5 if 384 well plates were used to print the microarray 9 0 if 96 well plates were used to print the microarray Custom to enable the edit boxes where you can enter values for custom pin spacing This forces the subarrays into straight rows and columns for quantitation The straightening does not display on the image bu
154. rrayExpress Microarray Analysis System 2 0 0 0 Temperature 1 5 LaserPower 1 1 LaserOnTime 5 5 Data record column headings Data Records GAL FILES The barcode symbols read from the image The origin of the image relative to the scan area the analysis Blocks relative to the scan area origin The version of the ScanArray Express software used to create the Results file The temperature of the scanner in volts The power of each laser in volts The laser on time for each laser in minutes Column titles for each measurement see below Extracted data Gene Array List files describe the size and position of blocks the layout of feature indicators in them and the names and identifiers of the printed substances associated with each feature indicator A 3 2 GPR Data Table A 4 below describes each column in the GPR file Table A 4 GPR Data Column Title Description Block Column Row Name Dia Technical Support 800 551 2121 or 617 350 9263 the block number of the feature the column number of the feature the row number of the feature the name of the feature derived from the Array List up to 40 characters long contained in quotation marks the unique identifier of the feature derived from the Array List up to 40 characters long contained in quotation marks the X coordinate in um of the center of the feature indicator associated with the feature where 0 0 is the top left of t
155. rrent image Header Information for Cy5 File name Image not saved to file File type Image File Scan date amp time 07 06 02 17 30 Microarray barcode 2393 8464 56 S 00743 JCNPH08 Scan protocol name Easy Scan Fluorophore Cys Laser power 100 PMT gain 75 Focus position pm 0 Scan resolution ym 20 Scan speed Full Scan area mm Top 00 Bottom 73 00 Left 00 Right 22 00 Instrument model Scan4rray Express Instrument serial number Unknown Client software version ScanArray Express Microarray Analysis System 1 0 0 0 Comments 4 Add comments if you wish and click OK 3 22 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Quant Chapter 3 3 4 5 Saving the Quantitation Results You can save quantitation results as a GPR or CSV file or as an Excel spreadsheet The GPR file does not save all of the columns if you want to save all of the data be sure to save it as a CSV file See Appendix A for a description of GPR and CSV files To save the results as a GPR or CSV file 1 On the Main Window click File in the Configure amp File group then click Save or Save As 2 In the dialog box that opens enter a file name and select the file type CSV or GPR Note To save the file in ArrayInformatics click File in the Configure amp File group and then click Save in ArrayInformatics If your system is not integrated with ArrayInformatics thi
156. rtion of Image In the Save Portion of Image window that opens use the mouse to select the portion of the image that you want to save Click OK In the Save Portion of Image dialog box that opens enter a filename and click Save To print an image 1 2 User Manual Click the tab of the image you want to print On the Main Window click File in the Configure amp File group then click Print pbtsupport perkinelmer com 3 7 Chapter 3 3 3 Using Easy Quant Using Easy Scan and Easy Quant The ScanArray Express remembers your settings from the last quantitation session or you can change them as you are ready to quantitate Scan the images to quantitate or open previously scanned images by clicking File in the Configure amp File group on the Main Window then clicking Open File To run Easy Quant 1 Inthe Main Window click Quantitate The Quantitate window opens select Run Easy Quant Quantitate Quantitation Template Adjust Template and Register Images Quantitation type Run a quantitation protocol Template From From From Array GAL File Specifications Informatics GAL file from which to read the gene list and spot statuses Bbc0032 HomeXchange S amples GeoS ample3 Quantitation method Fields for Adaptive circle Adaptive threshold Easy Q ua nt Fixed circle Histogram setti ngs Adaptive Circle Options Current options match defaults Normalization method L
157. rument Network Options The following window opens Network Instrument Settings x Instrument name EXPRESS Obtain IP address from DHCP Specify IP address IP address 10 0 Subnet mask 255 0 Default gateway 10 0 Cancel l Technical Support 800 551 2121 or 617 350 9263 ScanA rray Express System Settings User Manual Chapter 7 Select Obtain IP address from DHCP which allows the instrument s network settings to be determined dynamically at startup or Specify IP address which gives the instrument a fixed set of network settings Click OK to send the changes to the instrument The instrument must be restarted after the network settings have been changed The recommended way to restart the instrument is to use Restart the Instrument Software and OS from the Service Features menu pbtsupport perkinelmer com 7 17 Chapter 7 System Settings 7 18 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Diagnostics and Troubleshooting 8 1 Overview Chapter This chapter discusses diagnostic tools and basic troubleshooting and error recovery procedures that are available to help identify problems with the instrument 8 2 Diagnostic Tools ScanArray Express diagnostic tools include Start up Diagnostics In addition various LEDs on the instrument panel display the status of the instrument 8 2 1 Start up Diagnostics This diagnostic tool is executed automatically each
158. rument power to cause the instrument to re initialize Used exclusively by service personnel Using this feature prevents the user from being able to properly operate the instrument This is highly recommended to be used before shutting down the instrument Before turning off the instrument using the power switch click this button to observe the instrument s ready LED turn off wait approximately 30 seconds and then use the instrument s power switch to turn off the instrument pbtsupport perkinelmer com Chapter 7 Instrument Configuration Instrument model Instrument serial number Instrument server version Hardware device driver version 2 0 6 TCP IP address PAM hardware version PAM software version GIM hardware version GIM software version Hardware Options Option Status Barcode Reader Installed Arraylnformatics Installed Autoloader Not Installed Laserl Installed Laser2 Installed Laser3 Installed Laser4 Installed Laser5 Not Installed Filter1 Filter2 Filter3 Filter4 Filter Filter Filter Filters Filter3 Filter10 Filter11 Not Installed Filter12 Installed System Settings Figure 7 7 The Instrument Configuration Window from Save Instrument Configuration 7 6 2 Setting the Instrument Network Options 7 16 You can change the instrument name the name seen by the network and the network settings To change the network settings 1 Onthe Service Features menu click Set Inst
159. s button is dimmed and unavailable to use User Manual pbtsupport perkinelmer com 3 23 Chapter 3 3 24 Technical Support 800 551 2121 or 617 350 9263 Using Easy Scan and Easy Quant ScanArray ie Express Scanning with Protocols Chapter 4 Chapter Summary Overview 4 1 Running a Scan Protocol 4 1 Creating a Scan Protocol 4 4 The Scan Protocol Wizard 4 5 Using Scan Protocol Tools 4 15 Creating an Image Autosave Protocol 4 24 4 1 Overview A Scan Protocol lets you set up parameters for scanning including scan speed scan area fluorophores PMT Gain laser power and AutoSensitivity and save the parameters to be used again An Image Autosave Protocol is used to save the scanned images it specifies the folder file naming convention and file type to use when saving the images Protocols can also be defined in a Protocol Group allowing you to use multiple protocols to scan a single microarray A Batch Set allows you to run multiple protocols or protocol groups on to 20 slides in an autoloader See Chapter 6 Automating Scans and Quantitation for more information on protocol groups and batch sets This chapter provides instructions for running a Scan Protocol and for creating Scan Protocols and Image Autosave Protocols i gt Tip If you are a new or infrequent user you can use Easy Scan to become familiar with the scanning process before creating and using Scan Protocols See Chapter 3 for informa
160. saturation Lowering the PMT Gain or Laser Power setting will avoid this A formula that defines how the scanner hardware and Software execute the scanning portion of a microarray experiment A scanning protocol consisting of a set of parameters allows the user to define the parameters one time and then repeatedly execute the same protocol to scan multiple microarrays in an identical manner The dried remains of sample fluid printed onto a substrate Also called sub networks they are used to partition network addresses for efficiency and security Subnet masks work by masking some number of the less significant address bits on all of the workstations in the subnetwork The piece of glass upon which the array is printed These take the form of microscope slides which have chemically active coatings on the glass that allow the printed spots of sample to bind to the substrate Transmission Control Protocol Internet Protocol is the basic communication language or protocol of the internet It is also used in private networks called intranets and extranets TCP manages the disassembling of a message or file into smaller packets that are transmitted over the internet and received by a TCP layer that reassembles the packets to the original message The IP layer handles the address part of each packet so that it gets to the right destination ScanArray Express Term Definition Virtual Memory XYZ axis The concept of using hard
161. scanning and quantitation with a sample slide see Chapter 2 Getting Started Scan and Quantitate the Geometric Test Slide 5 2 Running a Quantitation Protocol User Manual Once a protocol is set up it is very easy to run After acquiring your image s by scanning or opening previously scanned images using the File menu under Configure amp File on the Main Window you select a quantitation protocol to use pbtsupport perkinelmer com 5 1 Chapter 5 Quantitating with Protocols To quantitate an image using a protocol 1 Click Quantitate on the Main Window The Quantitate window opens where you can choose a quantitation type Quantitate Quantitation Template Quantitation type C Run Easy Quant Quantitation protocol Easy Quant Adjust Template and Register Images Stat Cancel Figure 5 1 Selecting to Quantitate Using a Protocol 2 Select Run a quantitation protocol The settings for performing the quantitation are included in the quantitation protocol Unlike when using Easy Quant you cannot change the settings in this window 3 Select a protocol click the Click to Select button and select a protocol from the Select a Quantitation Protocol window that opens Click OK On the Quantitate window the button fills with the name of the selected protocol 4 Click Adjust the Template and Register Images to adjust the template to match the array and register the images if more than one image is displa
162. scent molecule um 20 scan lines second lt 2 5minutes for a 20 x 30 mm area at 10 microns 16 bit dynamic range over 4 orders of magnitude Both less than 5 CV TIFF BMP JPEG or Raw 8 user selectable rainbow gray red orange yellow green blue and purple Windows Pentium III 1 GHz processor 40 GB hard drive 512 MB RAM 19 color monitor CD RW 1024 x 768 x 65K color two 10 100 Mbs Ethernet network interface card NIC Windows 2000 SP1 Internet Explorer 5 0 or later Lite 30 L x 13 W x 14 H 75 cm x 32 cm x 36 cm Express 30 L x 16 W x 16 H 76 cm x 41 cm x 41 cm HT 38 L x 16 W x 16 H 97 cm x 41 cm x 41 cm 29 L x 13 W x 12 H 74 cm x 33 cm x 30 cm Appendix C ScanArray Specifications Specifications Description Weight Scanner External Laser Electrical ScanArray Instrument External Laser additional requirements Safety Agency Approvals Electrical Safety Laser Safety Electromagnetic Emissions Electromagnetic Immunity FCC Label for Class A Products ICES 003 Label for Class A Products Technical Support 800 551 2121 or 617 350 9263 Lite 70 Ibs 32 kg Express 82 lbs 37 kg HT 108 Ibs 50 kg 55 pounds 25 kg Autoselecting from 100 240 V 3 5 A 50 60 Hz Autoselecting from 100 240 V 15 A 50 60 Hz UL UL 3101 1 1993 Electrical Equipment for Laboratory Use USA amp Canada TUV EN 61010 1 A2 1995 Safety Requirements for Electrical Equipment
163. t 1 20 20 Slot status Full C Empty Ignored I Save in Arraylnformatics database util oes from all slots in the range I Set protocol group IV Set scan protocol IV Set image autosave protocol J Set quantitation protocol J Set sprdsht autosave protocol Protocol group Scan protocol Image autosave protocol Quantitation protocol Spreadsheet autosave protocol Name Name Name Name Dr Thomas Dr Dynamic Repeatability Sin Geo Slide 32x32 Demo Ger Geometric Ger Rotated Uniformity Sin Static Repeatability 4c 4 ois ot E is Protocol group description Protocol description Protocol description Protocol description Protocol description 16DyesStd Protocols beginning with cannot be executed with current hardware Protocol groups beginning with contain steps that cannot be executed with current hardware These steps will be skipped Cancel Figure 5 5 Range of Cassette Slots Window 2 Refer to the following table and set the parameters for the range of slots Item Description First Slot Enter the number of the first slot and the number of the last slot to Last slot include in the range Set slot state Check this box to set the status for all slots in the range Set use of Check this box to enable the selection box below it for saving in Arraylnformatics Arraylnformatics If your system is not integrated with Arraylnformatics this box is be dimmed and unavailable to select Save to Check this box
164. t affects the quantitation results This describes the position and size of each spot in a subarray Enter the number of rows and the number of columns of spots that are in each subarray Enter the horizontal and vertical spot spacing measuring from the center of a spot to the center of the spot in the next row or column Enter the spot diameter in um This forces the spots into alignment for quantitation The straightening does not show on the displayed image but affects the quantitation results 3 Click Completely Reset Template to the previous settings or click Modify Template Using Changes Only to save your changes 5 4 4 Quantitation and Normalization Method Specify the quantitation method and normalization method to be used by this protocol ScanArray Express uses the quantitation method to construct a pixel by pixel map that indicates the property of each pixel in the image Normalization corrects the intensity of each spot for variations in the overall intensity of the images with respect to the control image You can use the defaults Adaptive Circle quantitation method and LOWESS normalization Technical Support 800 551 2121 or 617 350 9263 ScanArray TM Express Quantitating with Protocols Chapter 5 To choose a quantitation method 1 Open the Quantitation and Normalization window Quantitation Protocol Composite Cy3 Contra cys 1 Se a ce 4 Quantitation and Normalization Quantitat
165. t statuses field fills with the name of the selected file See Defining a template Using a GAL File on page 5 8 From specifications Click this button to manually define the array pattern The Template Specification window opens where you enter the values See To define a template from specifications on page 5 9 User Manual pbtsupport perkinelmer com 5 7 Chapter 5 Item Quantitating with Protocols Description From Arraylnformatics Modify Template Dragging with Mouse Subarray Properties Zoom to Subarray Click this button to select template information from the Arraylnformatics database if your ScanArray Express system is integrated with Arraylnformatics Refer to the Microarray Laboratory Integration Guide that ships with the ArrayInformatics software If your system is not integrated with ArrayInformatics this button appears dimmed and is unavailable to select Select one of the behaviors for the mouse Moves or resizes all subarrays Rotates all subarrays Moves a subarray Opens a dialog box where you can enter the following parameters and then copy them to all subarrays Subarray top left corner x Subarray top left corner y Straighten spot rows and columns in this subarray Click to zoom the display on a subarray 3 Modify the template using the buttons in the Modify Template group 4 Click Next or click Quantitation and Normalization Method to go to the next window
166. te the home sensor and applies the appropriate calibration offset If an item fails the diagnostic test during Start up refer to Section 8 3 for troubleshooting tips Technical Support 800 551 2121 or 617 350 9263 ScanArray TM Ex press Diagnostics and Troubleshooting Chapter 8 8 2 2 Instrument Statistics Accumulation The following statistics are recorded and available for review Laser On time Total slides loaded unloaded 8 3 Troubleshooting 8 3 1 Hardware 8 3 1 1 Ready LED Flashes Yellow The instrument performs a variety of self tests of different modules upon initial power up If any one of them fails to initialize properly the ready light will flash yellow 1 Ifan external laser is present ensure the interlock block is installed on the rear panel of the instrument If it isn t power down the instrument install the interlock block and power up the instrument again 2 Ifyou do not get a steady green light after a minute or so try removing the front panel and putting it back on again This will reset the two safety interlock switches 3 Power down the instrument and power it up again If the Ready LED continues to flash yellow contact PerkinElmer Life Sciences 8 3 1 2 External Laser Will Not Turn On 1 Ensure the interlock on the rear panel of the external laser is plugged in If it isn t power down the instrument install the interlock block and power up the instrument again 2 Ifthe laser stil
167. teria Spot Status Spot Pixels To show or hide spots by their status 1 Click Show Hide Spots by Status Shown Spot Statuses Spots are displayed or excluded based on spot status Check the statuses that should be displayed ME V Not found IV Absent MV Good IV Bad Cancel l The spot status definitions are as follows Status Definition Found The spot was found within that position on the spot template Not found No spot was found within that position on the spot template Absent The spot was reported as missing within the GAL file Good The spot met the quality criteria Bad The spot did not meet the quality criteria 2 Check each status for which you want spots to display uncheck each status for which you want spots to be hidden 3 16 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Quant To show or hide columns of information Chapter 3 1 Click Show Hide Columns The Show Hide Columns dialog box displays Show Hide Columns Categories of columns Category Cy3 Cy5 Cy5 and Control Check the columns to show in the spreadsheet Subarray Location col row Spot Location col row Gene Name Gene ID No Spot Location x ymy pm Spot Status Sum of Median Spot Sum of Mean Spot Spot Size pixels Bkgnd pixels Diameter ym Footprint pixels gt Show
168. the spots Leave the template in a position with the upper left hand template circle near the upper left hand spot in the image To register the images click the Composite tab The images are not perfectly aligned if traces of individual color such as green and red are visible around the outside of one side of each spot Use the Zoom controls as desired to make it easier to view the registration Align the images by clicking the left right up or down arrows located at the left side of the window Each click on an arrow moves the image one pixel in the direction of the arrow Move the images until they are well registered that is many spots appear yellow Click OK Under Quantitation Method select Adaptive Circle Under Normalization Method select LOWESS Click Start The ScanArray Express finds the spots a progress bar displays showing the progress of finding spots and immediately begins quantitation 2 8 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Getting Started Scan and Quantitate the Geometric Test Slide Chapter 2 2 5 Viewing the Quantitation Results The quantitation results display in the Main Window as a Spreadsheet a Scatter Plot and a Distribution Plot each in a separate tab The data is linked in all views when you click a data point in one view the data point is already selected if you change to a different view gt ScanArray Express BBE Perki
169. the Properties button 3 4 3 Viewing the Distribution Plot The Distribution Plot is a simple yet powerful data visualization tool This feature allows you to select any type of quantitated data displayed in the spreadsheet and map it back to the spatial arrangement of the microarray using colors to indicate the value of the data The Distribution Plot is of particular value for diagnosing or troubleshooting microarray problems based on the idea that in a well processed array with a large number of spots there should be no significant spatial correlation of any property 3 20 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Using Easy Scan and Easy Quant User Manual Chapter 3 For example if ratio data were biased toward the green at the top of each subarray and biased toward red at the bottom of each subarray that would suggest a problem with attachment or hybridization Similarly if the spots in one subarray had diameters significantly smaller than the others that would suggest a problem with a pin The Distribution Plot allows you to see all of these phenomena and more with ease Run Scan Quantitate Configure amp File File Configure Spot Viewer Composite Median 4337 0 3 Median 41846 0 Spot Index 785 Gene name ACK Distibution Plot of Cy3 Ratio of Mean Spot EE Zoomin Zoom Gut Zoom Ful You can select a column of s
170. the protocol wizard 5 4 1 Basic Information In the Quantitate Protocol Basic Information window provide a name and description for the protocol To name a quantitation protocol 1 Fill in the information as listed in the following table Item Description Name Enter a name for the quantitation protocol so you can recall it easily You must enter a name Description Enter a description for the quantitation protocol 2 Click Next or click Image Registration to go to the next window If only one image is open the next window is Template 5 4 2 Image Registration If more than one image is open Image Registration automatically appears in the wizard as Step 2 If only one image is open this step and the associated window do not appear Registration is done from the Composite tab by moving the Source image so it registers to the Control image That is the Control image position is fixed and the Source image is moved relative to the Control Each image that needs to be registered has to be set as the Source image for registration User Manual pbtsupport perkinelmer com 5 5 Chapter 5 Quantitating with Protocols To register the images 1 Open the Image Registration window Quantitation Protocol Composite Cy3 Conto Cy5 2 Zoom in Zoom Out Zoom Full 2 Image Registration Image to shift or rotate Cy5 Image offset pixets 0 0 Rotate Image 180 set foyer eae Help and Dire
171. ther countries please contact your local PerkinElmer Life Sciences office for the exact terms of the warranty User Manual PBTsupport perkinelmer com Page 4 ScanArray Express Glossary Term Definition Accuracy Administrator Al Application Software Array Arraylnformatics HT Barcode Batch Set Bi directional Jitter BMP Calibration File User Manual Is the maximum positioning error that can occur between the commanded position of a motion control device and the actual position Accuracy is a measure of absolute error For example if the motion control device is commanded to move to position X1 Y1 Z1 but actually moves to position X2 Y2 Z2 the maximumdifference between corresponding values is the accuracy The person in your organization that manages the facility network and or sets up your Windows NT 2000 workstation See Array nformatics Software designed to perform a specific function directly for the user ScanArray s application software communicates with the embedded instrument software A collection of spots printed on a substrate arranged in a pattern of regular rows and columns A microarray gene expression database and visualization software available from PerkinElmer Arraylnformatics can share data with ScanArray Express systems that are configured for integration with ArrayInformatics An accessory device in scanner that uses a motor to automatically load substrates into
172. time the instrument is powered ON If the interlocks are open when the software is started diagnostics are performed after the interlocks are closed A system check is performed to ensure that all major functions are operational The status can be reviewed by clicking the Diagnostic tab in the Main Window Status is indicated using Pass Fail criteria Start up Diagnostic Test Purpose Attenuator Initialization Attenuator Homing Filter Wheel Initialization Filter Wheel Homing Objective Z axis Initialization Includes all initialization and homing of the attenuator Moves the hardware to accurately locate the home sensor and applies the appropriate calibration offset Includes all initialization and homing of the filter wheel Moves the hardware to accurately locate the home sensor and applies the appropriate calibration offset Includes all initialization and homing of Z axis hardware User Manual pbtsupport perkinelmer com 8 1 Chapter 8 8 2 Start up Diagnostic Test Diagnostics and Troubleshooting Purpose Objective Homing Servo Y axis Initialization Servo Homing Galvo Interface Module GIM Boot Galvo X axis Initialization Galvo Homing PMT Amplifier A D Module PAM Boot Laser Initialization Laser Communication Barcode Reader Initialization Barcode Reader Communication Autoloader Initialization Autoloader Program Download Autoloader Setup Autoloader E
173. tion on using Easy Scan For a tutorial that leads you through scanning and quantitation with a sample slide see Chapter 2 Getting Started Scan and Quantitate the Geometric Test Slide 4 2 Running a Scan Protocol User Manual A scan protocol is the most efficient and reliable method of scanning microarrays requiring the same scanning parameters With a user programmed protocol each user can scan microarray slides using the same parameters thus eliminating user to user variability from slide to slide pbtsupport perkinelmer com 4 1 Chapter 4 Scanning with Protocols To run a Scan Protocol 1 Click Sean on the Main Window The Scan window opens where you can choose a scan type Scan types on systems with an HT autoloader Scan Area cc Scan type Run Easy Scan Scan type Run a batch set Run a protocol group I Obtain scan protocol from barcode Scan protocol Select a Scan Protocol Obtain image autosave protocol from barcode Image autosave protocol Select an Image Autosave Protocol Fields for running Perform automatic quantitation a Scan Protocol Quantitation protocol Select a Quantitation Protocol J Spreadsheet autosave protocol Select a Spreadsheet Autosave Protocol J I Automatically save in Arraylnformatics To change the scan area select a different scan protocol Cancel Figure 4 1 Scan window 2 Select Run a scan protocol The fields for
174. tocol Dialog Box Note The items are added to the filename in the order that they were checked as shown in the File name pattern If you want to change the order uncheck all boxes then recheck them in the order that you want them to appear in the filename User Manual 5 Use the following table Item Description Name Enter a name for the image autosave protocol Use your own name or some other text that makes the protocol easy to find Description Enter a brief description Pattern for File Check the box for each item that you would like to include in the file Names names The selections include Fluorophore Barcode Date Time and Custom text If you check Custom text the text field becomes active and you can enter up to 64 characters of text To create a unique filename for each image you must include the fluorophore the date and the time File types Check each file type that you want to use TIFF is the preferred and default format for storing the output image Image information is not supplied with BMP or JPEG files pbtsupport perkinelmer com 4 25 Chapter 4 Scanning with Protocols Item Description Path to output files Enter the desired path for output files or use the Browse button to locate the path which can be a local path or a path on the network If a directory that doesn t exist is specified in the pathname a folder will be automatically created for it 6 Click OK to save the chan
175. total number of feature pixels the total number of background pixels the sum of the median intensities for each wavelength with the median background subtracted the sum of the arithmetic mean intensities for each wavelength with the median background subtracted log base 2 transform of the ratio of the medians the type of flag associated with a feature the normalization status of the feature included not included the median feature pixel intensity at wavelength 1 with the median background subtracted the median feature pixel intensity at wavelength 2 with the median background subtracted the mean feature pixel intensity at wavelength 1 with the median background subtracted the mean feature pixel intensity at wavelength 2 with the median background subtracted the signal to noise ratio at wavelength 1 defined by Mean Foreground 1 Mean Background 1 Standard deviation of Background 1 the sum of feature pixel intensities at wavelength 1 the number of the feature as it occurs on the array user defined feature data read from the GAL file Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Supported File Formats A 4 A 4 1 Appendix CSV Results File Format The CSV Results file contains sections enclosed by BEGIN SectionName and END SectionName Each section contains zero one or multiple rows between the BEGIN and END Each row contains data items separated by
176. tric Test Slide User Manual When finished your scanned image should match the images shown in this chapter To run Easy Scan on the Geometric Test Slide 1 2 Turn on the instrument and client workstation Start the ScanArray Express software Turn on lasers and 3 click the status button for each Insert the Geometric Test Slide into the instrument If you have an HT instrument with an autoloader insert the slide into the cassette align the slide using the alignment tool and insert the cassette into the instrument pbtsupport perkinelmer com 2 1 Chapter 2 Getting Started Scan and Quantitate the 5 Onthe Main Window click Scan The Scan window opens 6 Select Run Easy Scan The Scan window displays as shown below with the fields for Easy Scan settings Scan types on systems with an autoloader Scan Area Scan type Scan type EG 3 Run a scan protocol C Runa batch set Run a protocol group Scan resolution pm 65 810 Sw S0 850 Autoloader slot from which to load 1 20 1 PMT Laser Fluorophore Gain Power E Use Cyanine 3 55 30 M Use Cyanine 5 65 90 Easy Scan settings Scan Area Co ordinates Start position X mm 9 75 Area width mm 7 00 Start position Y mm 14 00 Area height mm 7 50 Set Scan Area to Full Microarray Show Zoom Window JE Automatically save image files locally To change the scan area drag I Automatically
177. tte chosen above Forboth simple and composite images For the composite image only use the rainbow palette for the simple image Contrast 0 Cancel Click the palette you want to use and select whether you want to use it for the current image and composite image or only for the composite image using the rainbow palette for all other images You can adjust the contrast if needed A Note Using the rainbow palette for the simple images provides more visual data by providing a spread of intensities instead of limited shades of just one color This allows you to differentiate more spots than you can with a single color Click OK Quantitating the Geometric Test Slide To quantitate the Geometric Test Slide Technical Support 800 551 2121 or 617 350 9263 To generate numerical quantitation data from the images quantitate the Geometric Test Slide scanned in the previous section using directions in this section With the two images generated in Section 2 2 still open click Quantitate on the Main Window The Quantitate window opens ScanArray ae Express Getting Started Scan and Quantitate the Geometric Test Slide Chapter 2 2 Select Run Easy Quant The Quantitate window displays fields for easy quantitation Use the settings in the following steps appropriate for the Geometric Test Slide Quantitate Quantitation Template Run a quantitation protocol From From From Array GAL File Specifications Inform
178. ttings Connection Scanning Quantitation Array Other i 2 Informatics Quality Measurement Settings Quality measurement method Footprint Signalto noise Signal to background Quality Measurement Parameters Maximum footprint um 100 Minimum signal to noise ratio 3 40 Lower limit Multiplier 2 Refer to the following table and enter your settings Item Description Quality These settings are used for the quantitation methods Measurement Settings Quality measurement method Quality Measurement Parameters Select a method to for quality measurement Formulas for methods are provided in Appendix D Footprint Signal to noise Lower limit and multiplier These fields are active or inactive depending on which Quality measurement method you selected 7 4 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Settings Chapter 7 7 2 4 Arraylnformatics Settings For ArrayInformatics users only the fields on this tab let you see which ArrayInformatics database the ScanArray Express is connected to and lets you specify the pathname for saving image files AN Note Changing the active database must be done using ArrayInformatics Database Tools installed on the ScanArray Express client computer For more information refer to the Integration Guide that ships with the ArrayInformatics software User Manual To set the Arraylnformatics Settings
179. uantitation Template adjusting 3 12 from GAL file 5 8 from specifications 5 9 Quick scan running 4 16 Ready LED flashes yellow 8 3 Safety agency approvals C 2 Sample size C 1 Scan resolution possible selections selecting Scan speed specification C 1 ScanArray Express exiting 1 9 main window 1 4 turning on 1 8 user interface 1 3 Scanning different ways to scan 1 6 Scanning field C 1 Scatter plot 2 10 sensitivity specification C 1 Service features 7 14 Size of instrument C 1 Size of instruments C 1 Source image changing 3 6 defined 3 6 Specifications excitation wavelengths C 1 specifications C 1 Spot template See Template Spreadsheet 2 9 3 15 Spreadsheet autosave protocol creating 5 19 System settings 7 1 Technical Support 1x Template adjusting 3 12 defining from GAL file 3 10 from specifications 3 11 registering images 3 14 Index Troubleshooting external laser 8 3 hardware 8 3 software 8 4 TIFF files 8 4 using diagnostic tools 8 1 yellow Ready LED 8 3 Tutorial quantitating the Geometric test slide 2 6 scanning the Geometric test slide 2 2 viewing and adjusting the images 2 3 viewing quantitation results 2 9 User interface 1 3 warranty 1 3 wavelengths C 1 weight C 2 Wizards quantitation protocol 5 4 scan protocol 4 5 workstation specifications C 1
180. ults automatically Checking this box enables the Spreadsheet autosave box where any configured protocols will display 3 When you are finished configuring the range click OK to save your changes and return to the Batch Set window Technical Support 800 551 2121 or 617 350 9263 ScanArray Express System Settings Chapter Chapter Summary Overview 7 1 ScanArray Express Settings 7 2 Configuring Fluorophores 7 7 Configuring Barcodes 7 9 Database Maintenance 7 12 Service Features 7 14 7 1 Overview User Manual ScanArray Express settings that are used frequently or those that affect the operation of the ScanArray Express system can be specified and stored using the Configure menu Settings include those for configuring fluorophores and barcodes and the system wide application settings such as network connection For systems that are integrated with ArrayInformatics the pathnames for automatically saving images and data to an ArraylInformatics database are specified here To access the system settings 1 Inthe Main Window click Configure in the Configure amp File group The Configure menu displays Configure Image l Autosave Quantitation l Spreadsheet Autosave Protocols Quantitation Protocols Automation Protocol Groups Batch Sets pareta Advanced settings covered in n this chapter Eluorophores Barcodes Database Maintenance pbtsupport perkinelmer com 7 1 Chapter 7 Syst
181. w Scan Area Move the mouse to the upper left corner of the area y Calibration Areas you would like to scan Press the left most mouse button down and hold it down Then move the y Calibration mouse to the lower right corner of the area you would i like to scan and release the mouse button To Modify the Current Scan Area Move the mouse over the edge that you would like to change The mouse pointer will change shape when you are close enough Press the left most mouse button down and hold it down Then move the mouse to where you would like the edge and release the mouse button Show Zoom Window Finish Cancel Figure 4 5 Scan Protocol Scan Area Window 2 Use the mouse to set the area to be scanned by drawing a rectangle of the scan area on the slide image or click Show Zoom Window A larger image allows you to select an area with greater accuracy Refer to the following table Item Description Microarray Diagram Displays the last scanned image or the resulting image if you ran a Quick Scan and lets you use the mouse to select an area to scan Full Microarray Automatically selects the entire microarray Use this button for 50 um scans to find the array Show Zoom window Opens a window with a larger diagram for easier selection of an area with the mouse and edit boxes where you can enter numerical scan area parameters instead Enter the coordinates for the Start position Area width and Area Hei
182. xels The button changes to Remove Filter so you can return to the previous appearance 3 Black threshold and full color threshold use these two slide controls together to adjust the image to minimize background noise and to increase the color for better visibility 4 Repeat for each tab you want to adjust To set palette options You can select a different palette or change the contrast The palette used in the composite tab cannot be changed it is always a blending of the palettes of the control and source experiment images or the rainbow palette If you open a palette when the Composite tab is selected the Composite Palette window that opens allows you only to adjust the contrast 1 Click Palette The Set Palette Options window opens Set Palette Options Name Palette Red Orange Yellow Blue Purple Gray Rainbow Use the palette chosen above For both simple and composite images amp For the composite image only use the rainbow palette for the simple image Contrast 0 ean 2 Click the color you want to use and select whether you want to use it for the current image or only for the composite image using the rainbow palette for all other images Adjust the contrast if needed A Note Using the rainbow palette for the simple images provides more visual data by providing a spread of intensities instead of limited shades of just one color This allows you to differentiate more spots than you can with
183. y the data record information name ID etc is included only for the first two spots ATF 1 0 8 5 Type GenePix ArrayList V1 0 BlockCount 4 BlockType 0 URL http www perkinelmer com microarray Supplier PerkinElmer Life Sciences ArrayersoftwareName SpotArray ArraySoftwareVersion 1 0 Blockl 400 400 100 24 175 5 175 Block2 4896 400 100 24 175 5 175 Block3 400 4896 100 24 175 5 175 Block4 4896 4896 100 24 175 5 175 Block Column Row Name Tp 1 1 1 VPS8 YALOO2W 1 2 il NTG1 YALO15C Technical Support 800 551 2121 or 617 350 9263 ScanArray Express User Manual Supported File Formats GPR Results File Format Appendix A GPR file is a Results file containing general information about image acquisition and analysis as well as the data extracted from each individual feature GPR Header A sample header file and a description of each entry is included in Table A 3 below Table A 3 GPR File Format Entry Description ATF 1 0 File type and version number 24 43 Number of optional header records and number of Type PerkinElmer results 2 0 DateTime 2000 02 09 17 15 48 Settings Geo Slide 32 x 32 Demo GalFile C Program Files PerkinElmer ScanArray Express Samples GeoSample32x32 gal Scanner Express Serial No 680065 Comment hyb 2673 PixelSize 10 ImageNAME 633 nm 543 nm FileNAME C Program Files PerkinElmer ScanArray Express S
184. yed See Easy Quantitation in Chapter 3 5 Click Start ScanArray Express finds the spots then immediately quantitates After quantitation the results display on the Main Window See Viewing Quantitation Results in Chapter 3 5 2 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Quantitating with Protocols Chapter 5 5 3 Creating a Quantitation Protocol To create a quantitation protocol 1 On the Main Window click Configure in the Configure amp File group In the Configure menu that opens click Quantitation Protocols Configure Configure amp File Eile Configure Spreadsheet l Autosave Protocols Quantitation Protocols Automation Protocol Groups Advanced Application Service Settings Features Eluorophores Barcodes Database Maintenance 2 The List of Quantitation Protocols window opens List of Quantitation Protocols Description Sort By Name Description 3 Sort the list optional by Name Description or Resolution User Manual pbtsupport perkinelmer com 5 3 Chapter 5 Quantitating with Protocols 4 Click one of the following in the List of Quantitation Protocols window to open the Quantitation Protocol wizard e Add to create a new protocol e Change after selecting a protocol to modify an existing protocol e Duplicate makes a duplicate of the highlighted protocol to help you create a new protocol from one that is alre
185. ys For the new or occasional user Easy Scan is easy to use all selectable settings are on one screen and you can change your settings as you work Use Easy Scan if Your lab uses one type of microarray and has one area of interest e You scan only one or two colors You want to walk up and use the equipment You can change settings as you work including the scan resolution the flourophores to be scanned PMT Gain and the area to scan 1 4 2 Scan Protocol A Scan Protocol uses pre defined settings that can be named saved and recalled to use again a protocol is useful when scanning different types of microarrays or when you want to use advanced features Using a scan protocol is simple once it is set up just load your microarray select the protocol and start Use a Scan Protocol if e You scan more than two colors or wish to scan any fluorophore multiple times Many users share the scanner and you want to automate file naming and saving for the users Your lab uses more than one type of microarray different image fields labeling protocols etc You wish to incorporate automation features into scanning such as Auto Sensitivity You want to automatically quantitate after scanning 1 4 3 Protocol Groups and Batch Scans Protocol Groups is a powerful new ScanArray Express feature Using a protocol group you can run different experiments on different areas of the same microarrays You can run seve
186. ystem log or event log keeps a record of all ScanArray Express activity in a log file In the file the ScanArray Express system logs the date and time of the activity the type and severity of the event the current user as logged onto Windows 2000 XP and a description of the event This log may be helpful in tracking operations or for use by Service personnel to diagnose any problems The system log tracks the following user requests to s start or stop a scan e add update and delete parameters modify system configuration start up and shut down the ScanArray Express application and tracks the following information changes in the status of client and instrument software communication software and hardware failures 8 4 1 Viewing the System Log 1 On the ScanArray Express Main Window click the Log tab 2 Use the scroll bar to move through the display as needed 8 4 Technical Support 800 551 2121 or 617 350 9263 ScanArray Express Supported File Formats Appa A Summary Overview F 1 Gene Array List gal Format F 1 GPR Results File Format F 5 A 1 Overview The ScanArray Express supports the following files which are described in the subsequent sections GAL An array content file is created by the SpotArray Microarray printer after a printing procedure This file can be called into ScanArray Express as a template for quantitating GPR A quantitation results file created by the ScanArray Ex

ScanArray Express Microarray Analysis System User Manual

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