Home

AIDA Microarray for Windows User's Manual

1. 2 IM MOMS i a GR 0 SU ie os a Tu cam tM eee eee tee ee Re et te Rte ee Figure 2 29 Schematic Display Window Example Alternatively the deviation of the spots to the mean value of data of interest can be coded by color Values excluded from the Schematic Display via the Exclude from schematic option of the Array Schematic toolbox empty dots guide dots or bad dots appear in gray and are marked with a cross to avoid confusion with real gray scale values To open the Schematic Display window e Choose View gt Schematic Display AIDA Microarray User s Manual 28 Because of the permanent linkage between all data windows you can use the Schematic Display window to easily navigate through the array on the Image window in order to select spots or spot groups E Aida Microarray E E i81 xl File Edit Evaluation Options View Window Help elele slal alel evaiuation 1 A RS 2 array Result Table c J Bl x Probe Scatter Plot c E 51 x Display Control Index Label Type Name ID x Position y Position Diameter Channel 635 w1 Y Data Normalized v Channel Display pixel pixel pixel IH E Ea 635 w1 77 83613 en ore
2. Figure 3 44 Selecting Repeat Pattern Option rra Label 9599 E 9601 02 24 9602 38P24a 9603 50 24 3604 02P24b 9605 DBP24a Figure 3 45 Hierarchic Labeling Example Repetition Pattern In this example 02 identifies all samples dots from microtitre plate number 02 P24 denotes the wells position on this microtitre plate from which the sample was taken Since in this example each sample was applied to the filter twice the suffixes a or b are used to identify the sample clones 104 AIDA Microarray User s Manual Global labeling ignores the level structure of the array and enumerates the spots according to a given scheme and running direction over the entire array Result Table Onco_ pm Inter Ps Figure 3 46 Global Labeling Example As shown in the screenshot above all rows and columns of the array are consecutively numbered The spot label in this example consists of only one two part ID number denoting the global absolute position of the spot in the overall array grid label 1 7 identifies the seventh spot in the first row As for hierarchic labeling the substructure of the identifier depends on the labeling scheme for the respective array level chosen from the pull down menus used to define labeling To set the labeling scheme for spots Click the Define Array Labeling button in the Grid Definition section of
3. Schematic Display Probe Table Options Schematic Histogram portrait Repetition Deviation fulltahle Analysis Table LII a Schematic Display Options portrait Analysis Table Options portrait full table C rows above a1 1 1 Figure 2 68 Print Microarray Dialog Using Microarray 59 Click the check boxes for the various items to enable or disable them Select the number of copies you want to print The margins of the printout can be selected in the Edges part of the dialog The Header button opens the following dialog Page Header and Footer X Header file path page number current date Footnote author last change time software version Figure 2 69 Page Header and Footer Dialog Here the Header and Footer of the report can be configured to contain various image and evaluation specific data The Parameter data overlays the Image Probe Table Array Scatter Plot Probe Scatter Plot Schematic Display Schematic Histogram Repetition Deviation and Array Analysis Table information can be selected in the left part of the Print Microarray dialog In the right part of the dialog Parameter and Image Options section you can select Whole Image or Window Content from the first drop down menu Choose Landscape or Portrait format from the second drop down menu Further the printing of Over
4. aee ES Eu Spot Level E E 5 E I Figure 3 80 Specifying Index Positions for the Hierarchic Positions Method Using the tabbed panel of the Import Name Table dialog Link 1 Link 2 Link 3 and Link 4 you can specify up to four source columns to be imported into your target table Furthermore the tabs allow you to enter a user defined name for the column header of the resulting target column In addition the cells of imported columns Name or ID for example can be hyperlinked to internal or external databases so you can search the linked databases for the name or ID by just clicking the respective cell in the Array Result Table Name and hyperlink of imported columns can adjusted via the Settings of command selectable from the shortcut menu of the respective column Link 1 Link 2 Link Link 4 coum Table Header Link http eww ncbi nlm nih qow entreziquer fcgi cmd sse Figure 3 81 Link Panel 6 Enter the number of the source table column you want to import into your target table using the Column spin box of the Link 7 tab Look up the column number in the source table display shown in the lower part of the dialog 132 AIDA Microarray User s Manual Note Entering the column number 0 in the Column spin box of a Link n tab indicates that no source column has been selected for import on this particular tab 7 Enter a name for the column to be displayed in the header of the imported co
5. Adjusting the alignment parameters using the Alignment controls of the Array Measurement toolbox Adjusting parts of the overlay for better fitting with the image To adjust parts of the overlay 1 Select the area of the overlay you want to adjust using the respective selection tool 2 Double click on the selected overlay area to switch to EDITING mode 3 Edit the overlay s fields block and or spot saccording to your needs In AIDA Microarray the following three different editing modes are available for array overlays Toggle between these modes by pressing the Space key 82 AIDA Microarray User s Manual Distorting Drag one of the squared handles attached to each of the four corner of the array overlay in any direction while holding down the mouse button This introduces trapeze shaped distortions 23 242 Ph a 03 Us 06 AU 08 D iL dd us 13 14 16 MM E a wal ben T m m m DC A i ROS Figure 3 9 Distorting Array Overlay Stretching and Compressing After switching to this editing mode the array overlay appears with squared handles attached to each corner and edge Dragging a corner handle stretches or compresses the array along the longitudinal and traverse axis simultaneously ft m gt n m o UE CENE SE i et p ZHE ke S UE m
6. 120 Data Range Normalization 4 122 Ratio Comparison SeLUTlgS c sais a e Re baila 123 Importing Name 128 4 Appendix 135 Formal Description of Background Subtraction 135 Global Grid Background Dots 135 Weighted Background Dots 135 Weighted Image REGIONS tote wie ew anes 136 Global Image 34 2644 2405564056 CLE e eS 136 Lowest and DOS vis A hc ss ORE 137 LocalDot 137 137 NOM SDOL UMS fe dq pir ER d E 157 Formal Description of Normalization 137 Normalisation by Reference 1 157 Normalisation by Data 139 4 AIDA Microarray User s Manual Introduction 1 amp Overview Array technology enormously speeds up the generation of new data from molecular biology experiments It is of growing importance for many fields in life science and 15 on the way to enter applications in medical diagnosis essential tool to benefit from microarray experiments is a powerfull software evaluation tool AIDA Microarr
7. 2027 T 10 D Y b is 136 AIDA Microarray User s Manual Lowest Grid Dots Let B b i 1 N denote the set of spots in a block with the lowest signal values Let Signal denote the Mean or Median value and SD the standard deviation of the pixel intensity values for a spot The lowest grid dot background for the spots in the block is calculated as follows _ 2 Signal bi Area bi Area bj d 3 SD b Area b D N95 Area b 13 Local Dot Ring For each spot a circular area ring around the spot is taken into account for the calculation of background of spot Neighbouring spot areas are clipped from the ring The Bkg value is either the Mean or Median of pixel intensities in this area and the BkgSD their standard deviation Local Grid Ring Around each block a circular area ring is taken into account for the calculation of the background for all spots in the block The Bkg value is either the Mean or the Median of pixel intensities in this area and the BkgSD their standard deviation Mode of Non Spot For each block in the array the non spot area is the block area without the spot areas The spot areas might be inflated to avoid overshining problems The Bkg value for all spots in the block is either the Mean or the Median of pixel intensities in the non spot area and the BkgSD their standard deviation Formal Description of Normalization Normaliz
8. area Mean Median Figure 3 56 Specifying Local Dot Ring A zone around the overlay spot can be defined where no background subtraction value 15 measured This is especially helpful if spots with high intensities radiate above the measurement spot To define the zone enter an appropriate value in the Inflate Dots spin box on the Settings panel of the Array Definition toolbox If the Adaptive Diameter Segmentation is enabled the spot diameter value can differ from spot to spot but since the Inflated Dots value is added to the Dia max value see the Alignment Settings section all inflated dots have the same diameter value With Ring Width a distance around the measurement spot and the zone excluded from background subtraction can be defined for determination of the background intensity The Mean or the Median value of all the pixels within the ring is subtracted from an individual spot 112 AIDA Microarray User s Manual Note that the inflated dot regions of the neighboring dots which intersect with the local dot ring do not enter into the background calculation Local Grid Ring The Mean or Median value of all the pixels in a zone around the area of the smallest grid level 1s taken for background subtraction As for the local dot ring method a zone around the overlay spot can be defined where no background subtraction value is measured This zone is specified using the Inflate Dots spin box If the Adaptive Di
9. Markby Color Label Figure 3 65 Normalisation Controls The Normalisation controls allow you to set values for the following parameters e Data This menu allows you to select which type of measurement value should be used for normalisation Settings Mormalisatian Data Method Integral Area Median Ap Integral Bkg wv Mean Bkg exc Median Bkg Markby Color Label Figure 3 66 Choosing Measurement Type for Normalization Performing Microarray Evaluations 119 Method For the comparability of the results of two arrays a normalization of individual result set is necessary AIDA Microarray offers two methods of normalization which can be selected from the Method menu of the Array Definition toolbox s Settings section Settings Mormalisation Data Mean Bkg Method Reference 1013 Mo Normalisation gt Reference Dots Data Range Area exclude bad dots MI rer sf Markby Color Label Figure 3 67 Selecting Normalization Method After the normalization process is completed additional columns Normalized Reference are inserted in the Array Result Table For details about this columns see the Array Result Table section of this user s manual Reference Dots Normalization Method Settings Mormalisation Data Mean Bkg Method Reference Dots Area Array Fields _ Area Menu Arithmetic Mea
10. To remove the mark from a spot choose the Bad Spot Deselection tool and click the spot you want the flag to be removed Use the Flag as Bad Spot button to turn previously selected spots into bad spots Clicking the Unflag Bad Spots button removes bad spot flagging from all currently selected spots To relate an error code to the flagging of the spots choose a code number 1 to 7 from the Error Code spin box The error code is displayed for each bad spot in the Quality row of the Array Result Table and the Analysis Table The OK Spot Selection tool is used to explicitly flag the integration value of a previously rated spots as reliable ok Just select the OK Spot Selection tool and click the spot you want to mark To switch back to the original rating mark of a previously OK marked spot choose the OK Spot Deselection tool and click the spot you want the flag to be removed 42 AIDA Microarray User s Manual Use the Flag as OK Spot button to explicitly turn previously selected spots into spots Clicking the Unflag OK Spots button removes OK spot flagging from all currently selected spots Final Calculation Button Selecting the Final Calculation button all remaining calculations required for integration background determination automatic rating and normalization are performed For further details see the Alignment Settings chapter below Array Schematic Toolbox With the Array Schematic too
11. raytest Je AIDA Microarray for Windows User s Manual Part Number 2004 07 1 1 2004 raytest Isotopenmefger te GmbH Benzstr 4 D 75334 Straubenhardt Germany rights reserved This product or document is protected by copyright and distributed under licenses restricting its use copying distribution and decompilation No part of this product or document may be reproduced in any form by any means without prior written authorization of raytest and its licensors if any RESTRICTED RIGHTS LEGEND Use duplication or disclosure by the government is subject to restrictions as set forth in subparagraph c 1 11 ofthe Rights in Technical Data and Computer Software clause at DFARS 252 227 7013 and FAR 52 227 19 The product described in this manual may be protected by one or more U S patents foreign patents or pending applications TRADEMARKS AIDA AIDA Microarray AIDA Array Metrix and AIDA Array Compare are trademarks or registered trademarks of raytest inthe United States and may be protected as trademark in other countries All other product service or company names mentioned herein are claimed as trademarks and trade names by their respective companies THIS PUBLICATION IS PROVIDED AS IS WITHOUT WARRANTY OF ANY KIND EITHER EXPRESS OR IMPLIED INCLUDING BUT NOT LIMITED TO THE IMPLIED WARRANTIES OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT THIS PUBLICATION COULD INCLUDE TE
12. The configuration remains valid unless it is explicitly changed by the user The automatic export settings are timestamped internally In addition you can add a name and an ID which are included in the Parameter pane of the export file together with the internal time stamp Using Microarray 67 1 Choose Options gt Automatic Export The Automatic Export Setting dialog appears E Automatic Export Setting 2004 04 29 adf E xj Setting Name select Setting Edit Setting Save Setting setting Stamp 20040217 153519 6197 Array Result Table Export an Save no Cancel Parameter Included YES Table Header Included yes List Delimiter tabulator Decimal Delimiter comma Setting for Report Columns Index Included in Export YES Decimal Digits 1 Figure 2 74 Automatic Export Setting Dialog The top pane of the dialog shows the current settings parameters whereas the second displays the current settings for the automatic export of the Array Result Table Analysis Table Probe Table Click on the toolbar to switch between the settings pages 68 AIDA Microarray User s Manual 2 Click Edit Settings The Edit Automatic Export Setting dialog opens imi Edit Automatic Export Setting 2004 04 29 adf Array Result Table E3 Export on Save No Parameter i List Delimiter Table Header Tabulator Decimal Delimiter Com
13. ja g index increase 1 Ban contro 1 background dot reference dot remove attributes spotting pattern cancel Figure 3 42 Defining Spotting Pattern Control dots are spots which are used for quality control negative controls Their values are collected and averaged over the entire array The resulting values are displayed in the Probe Table To ensure the correct collection of the control dot values always use the same index for corresponding dots Note If empty spots are defined they can be used for subtraction of a background in the Background Definition section of the Array Definition toolbox In addition you can specify entire background and or reference dot position patterns for sub grids which are applied to all sub grids of the array All flags set by the spotting pattern definition can be overwritten manually using the tools of the Array Definition toolbox If a repetition pattern 1s given additional values for the mean of the related spots as well as the absolute and relative deviation of this repeated dots appear in the Array Result Table Empty and Guide dots are not listed in the Probe Table 102 AIDA Microarray User s Manual Spot Labeling In addition to the various ways of labeling each level of your array see above AIDA Microarray provides two options for labeling spots hierarchic and global Hierarchic labeling respects the defined array levels and connects the
14. 001 002 TETTE les abc Cancel ABC Rows Column Sel 123 OK Cancel Figure 3 25 Setting Prefix 7 To specify the appropriate label ordering click on the icon in the right corner of the panel s heading 8 From the drop down menu that appears select the appropriate entry ete aga an a Rows Columns get AB C OK E Figure 3 26 Setting Label Ordering Specifying Field Repetition Pattern Repetitions are spots from the same probe They can be defined as repeated spots in a block repeated blocks in a field and repeated fields in the array In AIDA Microarray any number of repetitions on any level are possible Performing Microarray Evaluations 93 To add a new field repetition pattern 1 Click the Pattern Definition button on the Repetition Pattern Definition section Array Definition Grid Definition zno pattern Rows ac a Figure 3 27 Repetition Pattern Definition Section Selecting Pattern Definition Button Pattern Definition Button Columns The Select Field Pattern dialog appears 2 Click the Add button Select Field Pattern E Add cs biodi Remove Figure 3 28 Adding Field Pattern The Add Field Pattern dialog opens 94 AIDA Microarray User s Manual 3 Specify the repetition factor index and increment count using the repeated index and increase spin boxes respe
15. 145 8 8224 124 5 789 0 0 00 mooo 7348 1505 avefocseofiszraf 2592 1246 95 Fwepemejsma tso eer rosa C wejemeopeea 1270 sre 11695 1900 sa 12004 isis 92 146 Fwepmmopesa 2023 1230 s120 000 aretoko 200 err 155 7988 1305 000 9105 sas reno ena 000 rer e210 asjur nos 6488 1210 000 Lwepmero swea 1498 4283 0 195 9 1057 91 136 5 931 0 0 00 4 esee elelelelelelale e telae le lm 4 k Figure 2 9 Array Result Table Example To display the Array Result Table e Choose View gt Array Result Table Data Description The possible columns of the Array Result Table are the following Table 2 1 Array Result Table Columns Columns Short Description Spot Infomation Index Running table row number Field Field label potential reference column for name table import Signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set specified in the Image Attributes dialog 12 AIDA Microarray User s Manual Table 2 1 Array Res
16. Maximum 200 47 100 Automatic Cancel Counts Minimum Maximum 318 Logarithmic Automatic Figure 2 34 Data Scaling Dialog Here the Minimum and Maximum value of the x axis 532 W2 Mean Bkg QL and y axis Counts can be selected In addition you can enable automatic scaling of either axis separately and logarithmic representation for the y axis only by checking the respective option Logarithmic and Automatic below the Minimum and Maximum spin boxes Using Microarray 31 Repetition Deviation Window This data window shows a scatter diagram of the measurement values of a channel for all repetitions defined on the array iBl x m HEN HEN m Deviation B 10 9 T 5 4 3 2 1 0 1 2 535 1 Log Marmalized Probe Median Figure 2 35 Repetition Deviation Window To open the Repetiton Deviation window e Choose View gt Repetition Deviation The type of the measurement values and the channel for which the scatter diagram 15 generated can be selected using the Schematic Display panel of the Array Schematic toolbox To specify the type of the measurement values to be used for the scatter diagram On the Array Schematic toolbox select the desired data type from the Data menu of the Schematic Display panel If you select Integral Background Mean Median Integral Bkg Mean Bkg Median Bkg or Normalized for each spot the x axis
17. Optional Enter the missing information for example emission filter data in the corresponing fields 6 Click Open To open one or more single channel image files 1 Choose File gt Open 2 Select the image you want to load from the AIDA Data dialog and click Open 76 AIDA Microarray User s Manual 3 In the Compose Multi Channel Data Set dialog click Add Channel to add additional single channel images to the data set resulting in a multi channel data set im Compose Multi Channel Data Set Channel 1 Excitation Emission Color Bes Examples Simple Array ti es File Information m E Add Channel Remove Channel Figure 3 2 Compose Multi Channel Data Set Dialog 4 Choose the display color for each channel from the respective Color menus imi Compose Multi Channel Data Set E Channel 1 Excitation Emission Color EE samples Simple Array tit green Channel 2 citation Emission Color iv Bfe sExamples Simple Array B File Information 3E Add Channel Remove Channel Figure 3 3 Choosing Display Color for Channel 5 Optional Enter the missing information for example emission filter data in the corresponing fields 6 Click Open Step 2 Loading the Template In AIDA Microarray you can load either one of the built in pre defined array templates shipped with the application or import an previously saved
18. View gt Array Scatter Plot The measurement channel you want to plot against the master channel can be selected using the Channel menu of the Array Scatter Plot window The data type can be selected via the Data menu of the Array Scatter Plot window For a more detailed view of the plot the Array Scatter Plot window provides a zoom function To zoom the scatter plot 1 Click the Selection tool on the main toolbar kerk Figure 2 20 Selection Tool on Main Toolbar Using Microarray 23 a 4 m 5 3 532 v7 Normalized Figure 2 21 Zooming Array Scatter Plot 24 AIDA Microarray User s Manual Alternative procedure 1 Click the Zoom tool on the main toolbar Tree Figure 2 22 Zoom Tool on the Main Toolbar 2 Draw a rectangle around the area of the plot you want to see in more detail Figure 2 23 Zooming Array Scatter Plot Alternative Procedure To review the data values of dots shown in the Array Scatter Plot window click the dot The coordinates of the dot are highlighted together with all repetitions of the dot 535 1 Normalized 532 7 Normalized Figure 2 24 Reviewing Array Scatter Plot Values Using Microarray 23 Shortcut Menu of the Array Scatter Plot The shortcut menu of the Array Scatter Plot window provides you with two options Scales and Grid Array Scatter Plot c raytest Aida Image Analyzer dev 4 00 027 ioj x Ch
19. an upper and a lower expression threshold Max Ratio Setting a Max Ratio value the highest ratio value possible can be very useful if you want to export the comparison data to another program post processing these data e g for cluster analysis Since these programs usually accept numerical data only and since calculation of ratios of values within background range does not yield reasonable results Microarray sets the ratio value to Max Ratio if the ratio calculation results in infinity In the majority of cases it 1s reasonable to set the Max Ratio value to 100 Usually you will review the statistics of the comparison using the correlation plot displayed in the Array Scatter Plot window In the correlation plot the vertical and horizontal yellow lines indicate the background thresholds vertical line Master Cut Off horizontal line Client Cut Off whereas the lines shown in non yellow colors indicate the respective expression thresholds set by the Min Expression spin box on the Ratio Comparison panel of the Array Definition AIDA Microarray User s Manual toolbox Two diagonal yellow colored lines represent the Max Ratio value Max Ratio and 1 Max Ratio selectable via the Ratio Comparison panel of the Array Definition toolbox Array Scatter Plot c Program Files raytest Aida Image Analyzer 4 00 0232 amples E ml x Chanel Data 1 1 0 1 mq m 0 01 ir io 0 001 0 000
20. detected Bkg Deviation specifies a threshold for each channel in terms of multiples of the standard deviation of the background values In the scatter plot the spots whose Normalized values are below the threshold of both the measurement and the comparison channel respectively are displayed in yellow The threshold 15 calculated as follows On performing normalization using the selected normization method Normalized values as well as normalized background Performing Microarray Evaluations 123 values Bkg Normalized are calculated The mean and the standard deviation of the normalized background values for the spots are calculated If the measurement value is background corrected the threshold is defined by the selected multiple of the standard deviation If the measurement value is not background corrected the threshold value is defined by adding the mean of the normalized background values to the selected multiple of the standard deviation Bkg Multiples defines a threshold which requires that Normalized values must be above a selected multiple of the normalized background Normalized gt n Bkg Normalized to be regarded as detected This method 15 useful if the standard deviation of the background signals can be determined only badly or not at all and the Bkg Deviation method therefore is not applicable This is usally the case when you use one of the global methods for background determination Min Expression Defines
21. field 3 Miarmeatar Figure 3 39 Loading Spotting Pattern via Spotting Pattern Drop Down List 100 AIDA Microarray User s Manual Defining New Spotting Pattern If you click on the Pattern Definition button next to the Spotting Pattern drop down list the Select Spotting Pattern dialog opens allowing you to define a new spotting pattern or edit delete a previously saved spotting pattern ES Array Definition Grid Definition zno pattern Pattern Definition Button Emus Figure 3 40 Opening the Spotting Pattern List Dialog to Define a New Pattern To define a new pattern Click Add and then enter a name for the new pattern in the Name text box Select Spotting Pattern biodi EM Remove Figure 3 41 Selecting Spotting Pattern Dialog Position empty dots guide dots multiple dotting repetitions control dots background dots and reference dots by first clicking the appropriate button in the Pattern Definition section and subsequently clicking in the fields of the displayed basic array structure For Performing Microarray Evaluations 101 repetitions any number of repetitions can be set for the array sub structure using the repeated spin box Further to allow easy tracking of the repetitions you can assign an index and a spotting related number via the index and increase spin boxes Add Spotting Pattern B x Pattern Definition empty dot quide dot 3 4 repeated B
22. from the Analysis Table These types are Empty Dots Guide Dots Background Dots Reference Dots and Bad Quality Dots Exclude from Analysis Table Empty Dots Guide Dots Background Bots Reference Dots Bad Quality Dots Figure 2 56 Exclude From Analysis Table Section Filter Section This panel allows you to guide the automatic filtering process for analysis result values provided by AIDA Microarray Filter Edit Integral PSL 60 0 or Integralifrea 5 07 60 0 Figure 2 57 Filter Section With Rules Assigned The automatic result value filtering process is based on rules defined by the user There are no built in default filtering rules so unless you define a specific rule for filtering no automatic filtering 1s applied The filter to be specified consist of a list of conditions Alternatives concatenated by an OR operator An Alternative is a AND linked list of conditionals for evaluating the data values of a particular spot The filter rules you define are Permission rules Values that do not meet the conditions stated in the rules are blocked from being displayed in the Analysis Table window Using Microarray 49 To enter a rule statement 1 Click the Edit button on the Filter panel The Array Analysis Table Clauses dialog appears 2 On the Alternatives panel click Add This adds the phrase 1 Clause is to the Alternatives list panel 3 Then click the Add button on the Clauses pane
23. has the background attribute set or is marked as empty use empty dots option checked Let d spot Position spot Position b denote the Euclidean distance between a spot and the i background spot The weighted background for a spot is calculated as follows 2 Signal b Area b eno o Aw where Appendix 135 Do Area b 5 OR Note If all spots of an array have the same shape and diameter the area nor malization has no influence on the background calculation Weighted Image Regions Let Signal denote the Mean or Median value and S D the standard deviation of the pixel intensity values for a given region on the image Let rg 1 N denote the set of all background regions in the array Let spot Position spot Position rg denote the Euclidean distance between a spot and the it background region The weighted background for a spot is calculated as follows Signal rgi Area rgi spol BkgSD Y SD one P rgi 7 where Area rgi i spot 8 Global Image Regions Let Signal denote the Mean or Median value and S D the standard deviation of the pixel intensity values for a given region on the image Let rg 1 N denote the set of all background regions in the array The background for a spot is calculated as follows Signal rgi Area rgi S Areata 8
24. indicates the median of the repetitions in the probe for the selected measurement value whereas the y axis indicates the relative difference between the spot s measurement value and the median value of the probe 32 AIDA Microarray User s Manual If you select Log Normized or Log Ratio for each spot the x axis indicates the median of the repetitions in the probe for the selected measurement value whereas the y axis indicates the absolute difference between the spot s measurement value and the median value of the probe If you select Ratio for each spot the x axis indicates the ratio of the median values of the repetitions in the probe for the normalized values of both channels whereas the y axis indicates the relative difference between the spot s Ratio value and the ratio of the median Normalized values of the probe ration ni where n is the Normalized value of the measurement channel and n is the Normalized value of the master channel ratio m My where m is the median of the repetitions in the probe for the Normalized values of the mesurement channel and m is the median of the repetitions in the probe for the Normalized values of the master channel is indicated on the y axis ratio 15 On the x axis the m m indicated ratio ratio ratio To specify the channel for which you want to display the repetition deviation On the Array Schematic toolbox select the desired data
25. intensity values of the spot Background intensity per pixel of a spot see the Background Settings section of this manual for details Standard deviation of the pixel intensity values of the spot Standard deviation of the background pixels of the spot see the Background Settings section of this manual for details Background corrected integral value Background corrected mean value Background corrected median value Number of saturated pixels Homogeneity value see the Rating Settings section of this manual for details Median of the measurement values of all repetitions in a probe The measurement category used to calculate the median is the category which is used for normalisation Integral Mean Median Bkg Measurement value of the spot minus Probe Median Difference divided by Probe Median Quality flag Normalized measurement value of the spot see the Normalization Settings section of this manual for details Binary logarithm of the Normalized value Normalized background value of the spot Reference value used for normalization see the Normalization Settings section of this manual for details Signal unit and length unit units of measurement depending on the type ofthe imaging device used to obtain the image and the options set specified in the Image Attributes dialog 14 AIDA Microarray User s Manual Table 2 1 Array Result Table Columns Columns Short Description The following colu
26. oa EUER ERES 97 Defining a Block Matrix 1 98 Defining a Spot Matrix 1 99 SPONE x gr lates EE ee Gee ee We eh Se ties 100 Loading Predefined Spotting 100 Defining New Spotting 101 Spol Labe hne dans Raab EE bap Edad AN E wm eee ee 103 Contents 3 Dette Patdmeleks eo wa eS eg adu eRe A 107 Alignment Settings cee hh eee eee eee 107 Background CMe A eee oe UPS ERAI ced be BM eA 108 Global Background 1 109 Weighted Background 111 Grid Background 1 111 Lowest Grid eA ebd eds 111 Local Dot RAS oerte arie DOC Dp Ead a oC RA 112 Local rid Rin 2S e REC Eee eda eR 113 Mode OE NOS BOL opta ret ette oq ate eine ate baies 113 Global Image REsions vue ee hae Peon Re 114 Weighted Image 115 25 3 275 d LER a mus 116 Normalization Settings 119 Reference Dots Normalization
27. on the Protocol Print toolbar allows you to configure the layout setting for the Probe Scatter Plot window You can select the paper orientation Portrait or Landscape by clicking the appropriate radio button in the Format section The Graphic option in the Print section enables or suppresses the print output of the graphic displayed in the Probe Scatter Plot window Protocal Print Print Preview 3 Probe Scatter Plot Format Portrait C Landscape xr Fint Graphic Figure 2 63 Probe Scatter Plot Print Options 54 AIDA Microarray User s Manual Print Options for Schematic Display To configure the layout setting for the Schematic Display window choose the Schematic Display button provided by the Protocol Print toolbar As for Probe Scatter Plot you can select the paper orientation Portrait or Landscape by clicking the appropriate radio button in the Format section The only option in the Print section enables or suppresses the print output of the Schematic Display window Protocol Print Frint Preview Bchematic Display Format Portrait Landscape r Print L Graphic Figure 2 64 Schematic Display Print Options Using Microarray 55 Print Options for Schematic Histogram The Schematic Histogram button on the Protocol Print toolbar allows you to configure the layout setting for the Schematic Histogram window You can select the paper orientation Portrait or Landscape by clicki
28. san 00 e orama men wo wm e area so wo owe asia aol a 86 B2A14 m 532 w2 Channel Adjustment Orientation e t x 635 w1 635 1 Normalized Mean 0 1 532 W2 Normalized Mean 4 3456789101234 12345678901234 12345678901234 t Xe ees SRNR Re X d at 96 XXX em a t t heXeeeXevexXe ttt 6 6 6 ttt 4 t t 66 tX t tt t t Xl Xetetettetititttm tette tt tt t t Xt t ttt Quantum level x eX XXX t n tee
29. the Array 67 Importing Templates 4 224 94 e ERREUR eue oU S OD RN ee EORR ERR 71 Importing GAL 1 72 Importing Name Tables P pd ven fta a Sie s 73 Performing Microarray Evaluations 75 Standard Evaluation 75 Step T Leadine Image Pile a vc ARE RS 75 Step 2 Loading the Template a5 3 ed see dee Eder Ein Step 3 Adjusting the Template to 79 Step 4 Starting Automatic Alignment and Calculation 80 Step 5 Saving the Byalualiom E ed RUE obs au Peak rack 82 Manual Alignment Adjustments 82 Adjusting parts of the overlay for better fitting with the image 82 Adjusting the position of particular 4 87 Adjusting the alignment 1 88 Defining a New Templates 89 erba aes eee ae eee hoot abe eae 89 Defining a Field Matrix 91 Array D finition Setups RC ERE 91 Specifying Field Repetition 93 Pennine Borden lines 2255365 phere te kee eee
30. the Array Definition toolbox Figure 3 47 Opening the Array Labeling Dialog Performing Microarray Evaluations 105 The Array Labeling dialog appears allowing you to specify the labeling type Hierarchic Labels or Global Labels Array Labeling hierachic labels global labels Columns ABC ee ee cancel Figure 3 48 Array Labeling Dialog The option for global labels are the same as for array levels see above 106 AIDA Microarray User s Manual Setting Parameters In some cases you might want to adjust the default parameter settings for the alignment and or calculation process You can do this using the Settings panel of the Array Definition toolbox Choose the parameter set you want to change from the Settings drop down list Alignment Settings If you choose the Alignment menu item the appearance of the lower part of the toolbox changes revealing control for setting alignment parameters Settings Settings Alignment Alignment Z Local Positioning Local Positioning Segmentation Segmentation Adapted Diameter hl Diameter 0150cm o Diameter 0150em Dia 0200erm E e Dia min D cm 4 Figure 3 49 Alignment Controls With the Alignment controls you can set default parameter values for the automated alignment processes used to match the position of the defined array spots with the real position of the dots in the array This align
31. the Image window ls Paste Delete Select All Duplicate Show Image Map Figure 2 4 Shortcut Menu Image Window Just click the Selection tool on the main toolbar select the object you want to manipulate and then choose the appropriate command from the shortcut menu s a e Figure 2 5 Selection Tool on the Main Toolbar Select All selects all overlays present in the active image The Duplicate option duplicates the array overlay Using Microarray 9 Show Hide Image Map shows hides a navigator palette which let you see the entire image as well as the portion you re zoomed into indicated by a red viewing rectangle You can stay zoomed in and move easily to another section by dragging the red rectangle within the paletteto another area EZ C ERampless Examples Microarray oO x P Figure 2 6 Image Map Palette You can adjust the anchor position of the palette in the Image window by choosing the appropriate option from the shortcut menu of the palette k Hide Left Top Corner Right Top Corner w Left Bottom Corner Right Bottom Corner Figure 2 7 Shortcut Menu of the Image Map Palette 10 AIDA Microarray User s Manual Formatting Overlays You can specify the font and array level you want to apply to the label overlays using the Image Overlays option of the Options menu After selecting Image Overlays from the Options menu the Image Overlays dialog appears
32. the settings and click Save to confirm the action Selecting Automatic Export Settings 1 On the Automatic Export Setting dialog click Select Setting The Select Automatic Export Setting dialog appears E Select Automatic Export Setting 2004 04 29 adi X Cancel 20040501 22204 5716 Delete example 2004 04 29 Figure 2 77 Select Automatic Export Setting Dialog 2 Select a settings set and click Select to complete the action 70 AIDA Microarray User s Manual To delete a settings set 1 Select the set you want to delete in the list of the Select Automatic Export Setting dialog 2 Click Delete Importing Templates Array templates are stored with the program and can be imported using the Import option of the File menu or by clicking the Import Template button File Edit Evaluation Options View Window Open Strg O Save Strg 5 Save Os Close File Edit Evaluation Options view Close All New Evaluation Save Evaluation As k Export Spotter Template Print Mame Table Print Preview Printer Setup Information 1 c Examples Onco_c adF 2c Desktop test adf Sci Clontech Large Array adf Exit Figure 2 78 Importing Templates Using Microarray 71 Importing GAL Templates GAL GenePix Array List templates are generated by many micro array spotters A GAL template file contains the geometrical structural as well as probe information o
33. toolbar Evaluation Options View Display Control Publishing Layout Array Definition v Arrav Measurement Array Schematic Array Analysis Table Figure 2 37 Evaluation Menu Toolbar Correspondences Using Microarray 35 Array Definition Toolbox The Array Definition toolbox allows you to define high density array overlays To display the Array Definition toolbox On the main menu click Evaluation and then click Array Definition Alternatively you can select the Array Definition button on the toolbar ERI Array Definition Grid Definition ist i 1g 24 Grid Definition Section Selection Made b gas Selection Mode Section select all repetitions hide array overlay Settings Alignment Local Positioning Segmentation Adapted Diameter Settings Section Diameter 0150cm e Dia Uzn em e Dia min 001 0 eo Figure 2 38 Array Definition Toolbox 36 AIDA Microarray User s Manual Grid Definition Section In the Grid Definition section of the toolbox you can define new array levels or edit existing ones Grid Definition Name Table Import Button Define Array Labeling Button Edit Array Spots of Selected Blocks Button Edit Array Blocks of Selected Fields Button Edit Array Fields Button Figure 2 39 Grid Definition Section Overview Note In Array Easy the functionality of the Grid Definition panel is restricted to e
34. type from the Channel menu of the Schematic Display panel Using Microarray 33 Analysis Table In contrast to the Array Result Table which lists the analysis results for the entire array the Analysis Table is used to display analysis results for a subset of the array Analysis Table raytest Aida Image Analyzer dev 4 00 023XEx Oo pizel 2 1 2 1 2 1 Refi 277 0 3053 0 634 6 404 6 290 0 409 0 52 ejes omen 20afoo50 o172 as ana poor o jaeno on _ Figure 2 36 Analysis Table Window Example You can define those subsets in two different ways manually or by filtering using user defined rules for details see the Array Analysis Table Toolbox section of this manual To open the Analysis Table e Choose View gt Analysis Table The Analysis Table window provides the same set of display controls as the Array Result Table window so please refer to the respective description in the Array Result Table section for further details 34 AIDA Microarray User s Manual Additional Toolboxes in Microarray After selecting the Microarray evaluation mode the standard AIDA toolbox set Display Control and Publishing Layout is expanded by four additional toolboxes Array Definition Array Measurement Array Schematic Array Analysis Table You can access these four additional toolboxes via the Evaluation menu or by clicking the appropriate button on the
35. user defined template To load a predefined array template 1 Click the Import Array Template button in the Template section of the Array Measurement toolbox Performing Microarray Evaluations 77 This changes the appearance of the Template section allowing you to select array templates from the template library 2 Select an array template and click OK Hen Array Measurement Template Rn Array Measurement Select Array Template ulation Lab Arraytor 500 1 Lab Arraytor 1 2 1 2 Lab Arraytar 500 2 Lab Arraytor 14 14 Clontech Atlas Large Array Clontect Atlas 1 2 Arra Research Genetics OK Cancel Figure 3 4 Selecting Array Template The resulting array overlay appears in red indicating the array overlay s EDIT status In addition the appearance of the toolbox section changes again revealing spin boxes for fine tuning the adjustment of the array template in terms of array overlay x and y offset image and array resolution as well as spot diameter see Step 3 Adjusting the Template to the Image for details To load a previously saved user defined array template 78 1 Choose File gt Import gt Template or click the Import Template button on the main toolbar Choose the template file you want to import from the file list in the Import Template dialog Now all data settings and overlays from the array template file are imported to the image data The overlays in the array templa
36. 1 Threshold m 3 0 Std Dev 1 05 ui 0 0001 0 001 0 01 0 1 1 532 7 Normalized Min Expression Figure 3 71 Scatter Plot Representation of Comparison Parameter The background threshold divides the scatter plot into four parts 1 Master value Master Cut Off and client value lt Client Cut Off Both master and client value are within the background range i e their ratio cannot be determined In this case Microarray sets the ratio value to 1 expression value No 2 Master value lt Master Cut Off and client value gt Client Cut Off In this case only the master value 1s within the background range whereas the client value is above the background threshold Their ratio cannot be determined but since client s measurement value can be determined in contrast to case 1 above the ratio value is set to the predefined constant Max Ratio expression value On Performing Microarray Evaluations 125 3 Master value gt Master Cut Off client value Client Cut Off This is the opposite case of 2 with the value of the master array above the background threshold and the client array value indeterminable The ratio value is set to Max Ratio expression value Off 4 Master value gt Master Cut Off and client value gt Client Cut Off Both values are above the background thresholds so their ratio is determinable client value master value If ratio value gt 1 the expression value is set to ratio val
37. Analysis Table Figure 2 1 View Menu Image Window in Microarray data are evaluated from the Image window It displays the different channels of a multi channel image in a single view EZ C Examples Examples Microarray m gt Figure 2 2 Image Window Example Using the Channel Display controls of the Display Control toolbox you can specify how the channels are displayed in the Image windows Select the Overlaid in false colors Tile horizontal or Tile vertical button to show all channels superimposed on each other in false color display display each channel in a different window pane side by side or on top of each other respectively E 3 Display Control Channel Display Overlaid in False Colors button ___ m ea D E Tile Horizontal button 635 w1 ia 532 2 Tile Vertical button Channel Selectors Figure 2 3 Channel Display Buttons of the Display Control Toolbox You can show hide channels using the channel selectors on the Channel Display panel 8 AIDA Microarray User s Manual Although the Image window might not always be on top it is always open The title bar of the Image window shows the full file path of the image To copy the content of the Image window to the clipboard select the Copy Content option from the Edit menu Shortcut Menu of the Image Window Cut Copy Paste and Delete are used to manipulate overlays for example array overlays text and arrows on
38. CHNICAL INACCURACIES OR TYPOGRAPHICAL ERRORS CHANGES ARE PERIODICALLY ADDED TO THE INFORMATION HEREIN THESE CHANGES WILL BE INCORPORATED IN NEW EDITIONS OF THE PUBLICATION raytest MAY MAKEIMPROVEMENTS AND OR CHANGES IN THE PRODUCT S AND OR THE PROGRAMS S DESCRIBED IN THIS PUBLICATION AT ANY TIME Contents 1 Introduction 5 eee i 5 Stare tod a La EH ears ERG Fee wee eRe Fee ees 6 2 Using Microarray 7 Data Windows in Microarray 7 Image Window in 8 Shortcut Menu of the Image 9 Formatting Overlays s dud d eri esl ye dee edel E es 11 Array Result Tables 6 e S Ede ren 12 Pata D escripiOD usns does dont deo deo Do died ndo e italie 12 Shortcut Menu of the Array Result 16 PHOS able ied orta ah at wea esca Rub S tuac RE 20 Data Descriptio RR EX De Rt Ref di dc da PS 21 Armay Sealer 25s borde doe dox uem pem dudas 23 Shortcut Menu of the Array Scatter 1 26 PODS SCANT PIO 22332 cud verd icto em d en dob dus 27 Schematic Display 28 Schematic Histogram Window 0 ccc eee eee es 30 Shortcut
39. Click Save to confirm the action 62 AIDA Microarray User s Manual The Array Result Table Export Options dialog appears allowing you to set further option for the export of the array results imi Array Result Table Export Options P x Table Header Cancel All Rows C Selected Rows Cancel f All Columns C Selected Columns List Delimiter Decimal Delimiter Tabulator C Point Comma C Comma C Semicolon System Detaults Figure 2 71 Array Result Table Export Options Dialog You can specify the Range of the exported rows and columns as well as the List Delimiter and Decimal Delimiter in the respective panels of the dialog 4 Complete the action by clicking the OK button The data can be saved to a simple ASCII text or MS Excel file Exporting Probe Tables To export the Probe Table 1 Choose File gt Export gt Probe Table 2 In the Probe Table Export dialog that appears enter a name for the export file 3 Click Save to confirm the action Using Microarray 63 The Probe Table Export Options dialog appears allowing you to set further option for the export of the array results imi Probe Table Export Options B X Table Header Cancel All Rows Selected Rows f All Columns f Selected Columns List Delimiter Decimal Delimiter Tabulator C Point Comma C Comma C Semicolon System Defaults Figure 2 72 Probe Table Export Options Dia
40. E ME 35127 jpg agito me Hm deut 3 1 E fiu i it im 14 15 16 17 18 19 20 EL 22 23 Figure 3 10 Stretching Compressing Array Overlay X Y Axis Performing Microarray Evaluations 83 Dragging edge handle stretches or compresses the array overlay in direction only 14 12 13 14 15 16 17 18 18 20 20 22 23 24 Qj 02 03 Q4 05 06 07 08 09 v leas ty tme etes usi iun BRUM NEL 4 4 TNT 2 1 LI IM FE TT EEE RE S REFER og legge es HL ALLELE EFEETEE tt 1888 8885 8854 L HERE EREE IL EREET SERERE TERE Stretching Compressing Array Overlay Y Axis Only ESEE 0 na 09 Figure 3 11 P nc Fs nempe aii Rotating Array Overlays Switching to this mode changes the six squared handles of the previous mode to circle shaped ones Dragging a corner circle rotates the array overlay around the opposite corner ee a LI oe ES Eg gags uu m ww ER IG H l J L M N o P tet ee ES Be o EE Lt s d Figure 3 12 Rotating Array Overlay Around the Lower Left Hand Corner 84 AIDA Microarray User s Manual Dragging edge circle rotates the overlay around its center Figure 3 13 Rotating Array Overlay Aro
41. Menu of the Schematic Histogram Window 31 Repetition Deviation Window 32 Analysis Tapie amp ite one Verh Vos ad ebd PE BU Eds Rel ete ta aad e dat 34 Additional Toolboxes in 35 Array Definition 36 Grid Definition Section ou oce ed oe Ghia pt de qnas ieee Mas drea d 37 Selection Mode 38 Se CUON a5 O43 UE RR dett e a 38 Array Measurement Toolbox 39 Template SecHol 40 selection Mode 40 SCCUON sy cue va Cd vex RA deu E VS 41 Rating Dot Values 42 Final Calculation Button ee eee eens 43 Array Schematic 43 Exclude From Schematic Section 44 Actual Probe 45 schematic Display eer eee Ee a s 46 Array Analysis Table Toolbox 48 Apply Selected Dots 5 48 Exclude From Analysis Table 49 Pi
42. adjusted and cells can be linked to external database by the user via column shortcut menu command ID number of the probe to which the spot belongs column name can be adjusted and cells can be linked to external database by the user via column shortcut menu command User definable columns for name table import purpose column name can be adjusted and cells can be linked to external database by the user via column shortcut menu command Signal unit and length unit units of measurement depending on the type ofthe imaging device used to obtain the image and the options set specified in the Image Attributes dialog Using Microarray 13 Table 2 1 Columns unused 2 gt Array Result Table Columns Short Description User definable columns for name table import purpose column name can be adjusted and cells can be linked to external database by the user via column shortcut menu command Spot measurement values listed for each channel Integral signal unit Mean signal unit Median signal unit Bkg signal unit Integral SD Bkg SD Integral Bkg signal unit Mean Bkg signal unit Median Bkg signal unit Saturated pixel Homogeneity Probe Median signal unit Difference signal unit Rel Difference Quality Normalized Log Normalized Bkg Normalized Reference signal unit Sum of the pixel intensity values of the spot Mean of the pixel intensity values of the spot Median of the pixel
43. allowing you to select the level of array Label Array by font font size font style regular bold for labeling E Image Overlays NR_ 3_XL TIF E X EH Array Overlay Label Array by C 1 Level 2 Level Cancel C 3 Level Spotting Fort Arial Size 1 1 1 print mark background regions overlay dots with bkg level Figure 2 8 Image Overlays Dialog In addition you can show or hide the regions used for background calculation by selecting the mark background regions check box Checking this option also sets marks for the local dot and grid ring as well as for mode of none spot if the respective modes are selected Further the options shows or hides the background image regions if the Global Image Regions or Weighted Image Regions background method is chosen for details about background definition see the Background chapter below If you check the overlay dots with bkg level option each dot is overlaid by its background gray level Using Microarray 11 Array Result Table This window shows the results of the array analysis in tabular form It summarizes the measurements of all channels in a single data view Values of multiple channels are listed in numbered child columns of a parent column Array Result Table c5 rayEest Aida Image Analyzer dev 4 00 022 Exa 10 Area 2 ualt Mean GL Median GL Bkg GL pixel 1 ey 1 2 1 2 1 315 2064 0 19874 0
44. alog appears offering you a list of available evaluation modes The list entries reflect the installed AIDA application modules lee Change Evaluation Mode Available Evaluation Modes 1D Densitometry 1D Densitometr GLFP 1D Evaluatian 1D Evaluation Cancel 1D Runlength Determination 1D Runlength Determinatian 1D Thin Layer Chromatography GLP 20 Densitometry 20 Densitometr 20 Thin Layer Chromatography GLP Annotation Array Matrix Crystallography dey Crystallography Material Science Microarray Autoradiography GLP Figure 1 2 Change Evaluation Mode Dialog 2 Select the Microarray list item and click OK Note The Microarray evaluation mode is only available if you purchased the AIDA Microarray module 6 AIDA Microarray User s Manual Using Microarray Data Windows in Microarray In addition to the AIDA standard data windows Image and Histogram the following data windows are available in the Microarray evaluation mode Array Result Table Probe Table Array Scatter Plot Probe Scatter Plot Duplicate Correlation Schematic Display Schematic Histogram Repetition Deviation Analysis Table To access these data windows choose the appropriate entry from the View menu view Window Help Image Histogram Array Result Table Probe Table Array Scatter Plot Probe Scatter Plot Schematic Display Schematic Histogram Repetition Deviation
45. alues of all the regions defined are averaged ori Eye pom goi ioc ea Gh noch oH Global Image Regions bg area Meant Median Figure 3 59 Specifying Global Image Regions 114 AIDA Microarray User s Manual Weighted Image Regions As for Global Image Regions arbitrary rectangles can be defined which you can use for weighted background subtraction for differences between global and weighted background subtraction see the Global and Weighted Background Dots section above irl ir pow gi az Echo bench cH DET g m nm F Weighted Image Regions 1 area Meant Median Figure 3 60 Specifying Weighted Image Regions It is strongly recommended that you define as many regions as possible to enhance background subtraction Also distribute the regions over the array as evenly as possible If this is not possible try to position the regions all around the respective array Performing Microarray Evaluations 115 Rating Settings If you choose the Rating menu item the appearance of the lower part of the toolbox changes revealing control for setting rating parameters Settings Fating Pixel Saturation 1 w Pixel Saturation Repetition Deviation Dok Homogeneity Fare satur
46. ameter Segmentation is enabled the spot diameter value can differ from spot to spot but since the Inflated Dots value is added to the Dia max value see the Alignment Settings section all inflated dots have the same diameter value spots of one sub grid are treated by the same background subtraction Inflate Dots O01 0 Ring width area Mean Median Figure 3 57 Specifying Local Grid Ring Mode of Non Spot In Mode of Non Spot the Mean or Median value of all the pixels in an area within the smallest sub grid around the measurement spots excluding the spots s pixels 15 taken for background subtraction With Inflate Dots a zone around each spot where no background is determined can be defined If the Adaptive Diameter Segmentation is enabled the spot diameter value can differ from spot to spot but since the Inflated Dots value is added to the Dia max value see the Alignment Settings section all inflated dots have the same diameter value Performing Microarray Evaluations 113 Inflate Dots O01 0 area C Mean Median Figure 3 58 Mode of Non Spot Global Image Regions Arbitrary rectangles can be defined which are used globally for background subtraction This done by averaging the Mean or the Median values of all the pixels within the arbitrary rectangle and applying the resulting value to all dots If you define more than one background region the v
47. annel 635 w 1 Data Normalized Scales Grid 0 001 535 1 Normalized ce 0 0001 1 05 0 0001 0 001 0 01 0 1 1 532 v7 Normalized Figure 2 25 Array Scatter Plot Window Shortcut Menu The Scales option allows you to configure the graphical output After selecting the Scales option the Date Scaling dialog opens Data Scaling E x 532 v7 Normalized Minirmurn M axirmurm 000042 3 14 Logarithmic M Automatic Cancel 535 1 Normalized Minimum 000011 E 8 85 Logarithmic v Automatic Figure 2 26 Data Scaling Dialog Here the Minimum and Maximum value of the x axis 532 W2 Normalized and y axis 635 W1 Normalized can be selected In addition you can enable logarithmic representation and automatic scaling of either axis separately by checking the respective option Logarithmic and Automatic below the Minimum and Maximum spin boxes 26 AIDA Microarray User s Manual Choosing the Grid option opens the Grid dialog box which allows you to lay grids over both axis that are an extension of either the big or the small ticks on the axes Harizantal Mone C Big Tics C Small Tics Cancel Help Mone Big Tics Small Tics Figure 2 27 Grid Dialog Box Probe Scatter Plot In the Probe Scatter Plot window the probe data the averaged values of the spots of a probe of the measurement c
48. ated BIXE Saturated Area 95 Figure 3 61 Rating Controls The Rating controls allow you to set values for the following parameters for the automatic rating process Pixel Saturation Check rate saturated pixel to enable the rating of saturated pixels Enter a threshold percentage of saturated pixels per area If the number of saturated pixels per area exceeds this threshold the spot is marked as bad and tagged with the label sat for saturated in the Quality column of the result tables Settings Pixel Saturation Iv rate saturated pixel Saturated Area 95 Figure 3 62 Pixel Saturation Controls 116 AIDA Microarray User s Manual Dot Homogeneity AIDA Microarray provides a automatic signal intensity homogeneity check function for spots The check is performed by measuring the signal intensity distribution within individual spots The Diameter spin box is used to specify the spot area in percentage of the measurement spot to which the check function 15 applied Using the Deviation gt spin box you can set a percentage of deviation from homogeneous intensity distribution To set a threshold intensity in multiples of background specify the appropriate value in the Integral gt spin box To activate the rating of dot deviations select the rate dot deviation checkbox Spots identified by this function are flagged sd strong deviation in the Quality row of the Array Result Table Analysis Ta
49. ation by Reference Dots Let Signal denote the measurement value of a spot which is used for the normal ization Let R rj 1 1 denote the set of all reference dots within the entire array a field or a block Appendix 137 For any spot within the entire array field or block respectively the Normalized value is calculated by Arithmetic Mean 1 C N Signal ri 14 Reference spot C 15 Normalized spot _ 0 16 Geometric Mean C exp x log Signal n 17 Reference spot C 18 Signal spot Normalized spot Variance Stabilization The Variance Stabilisation Normalization for microar ray data was proposed by Wolfgang Huber et al 2002 in the paper Variance stabilization applied to microarra data calibration and the quantification of differ ential expression This normalization method requires two or more channels which are normalized simultaneously The parameter estimation in AIDA follows the same robust max imum likelihood estimation which is mentioned in the above papers In fact in AIDA the optim function with method L BFGS B of the statistical R package is integrated In the current version of AIDA only the parameter estimation step for known not regulated genes of the Variance Stabilisation Normalisation is im plemented The papers include a second iteration step for selecting not regulated genes which is not implemented yet Inst
50. ay is designed for the fast and reliable evaluation of quantitative and qualitative data from all kinds of array experiments in life science It provides the optimized solution for slide evaluation with two or more fluorescence labeled targets Two to six channels can be evaluated simultaneously sharing the same highly efficient spot alignment algorithm and the automatic adaptation of the spot diameter With four simple steps precise data from array experiments can be generated 1 Load Image 2 Load Template 3 Alignment amp Calculation 4 Save and Export Beside this ease of operation AIDA Microarray has an unmatched flexibility in the adaptation to different array designs Spotting patterns can be defined with multiple spotting probes and control probes A great variety of background subtraction and normalization methods are available The Rating of Spot Value function allows you to check the quality of the array results manually as well as automatically The results can be displayed using various data view options including e Schematic Histogram view e Schematic Display view e Scattered Plot view In addition results can be filtered automatically saved exported to databases or transferred for further processing to the AIDA Array Clusterer software tool Starting Microarray To select the Microarray evaluation mode 1 Click the Evaluation button in the toolbar Figure 1 1 Evaluation Button on the Main Toolbar A di
51. ble and the dot deviation value 15 listed in the Homogeneity column Settings Diameter 100 0 Iv rate dot deviation Deviation gt 25 0 Integral gt 5 Bkg Figure 3 63 Dot Homogeneity Controls Repetition Deviation This options allow you to flag dot repetitions which deviate from the average value of the measurement type selected for normalization Checking the above probe median and or below probe median options enables flagging of deviating dot repetitions above and or below probe median To set the percentage of deviation from the median use the Deviation spin box and to set a Performing Microarray Evaluations 117 threshold for the median value given in multiples of the background specify the appropriate value in the Median spin box This restricts the application of the dot repetition flagging function to spots which are substantially above background Settings Flag Dot Repetition above probe median below probe median Deviation gt 25 0 Median HL 5 Bkg Figure 3 64 Repetition Deviation Controls 118 AIDA Microarray User s Manual Normalization Settings If you choose the Normalisation menu item the appearance of the lower part of the toolbox changes revealing control for setting normalisation parameters Settings Data Mean Bkg Method Reference Dots Area Array Fields Arithmetic Mean exclude bad dots MI rer rds
52. ch spin box Schematic Display Schematic Display Data Mean Bk Data Mean Bk E Channel 532 7 Channel 532 v2 Data Colors 52 Mean 50 Colors 52 Pich T Hj 376148 2 4 0 59 141 0 42 0 38 0089 15 876 8 18741 3 2 20 42554 9304 5 4 0 25 3875 aL Figure 2 53 Setting Data Range left and Specifying Step Size right Using Microarray 47 Array Analysis Table Toolbox With the Array Analysis Table toolbox you can configure the settings for the Analysis Table window E Array Analysis Table Apply Selected Dots Insert selected dots OEC Apply Selected Dots Section attributes Clear applied Exclude from Analysis Table Empty Dots Guide Dots Background Dots Exclude From Analysis Table Section Reference Dots Bad Quality Dots Filter Edit Filter Section View Analysis Table Figure 2 54 Array Analysis Table Toolbox Overview Apply Selected Dots Section The controls of this panel are used to manually add values to or remove values from the Analysis Table Apply Selected Dots Insert selected dots Remove selected dots lear applied attributes Figure 2 55 Apply Selected Dots Section 48 AIDA Microarray User s Manual Exclude From Analysis Table Section In the Exclude from Analysis Table section of the Array Analysis Table toolbox you can select some dot types for exclusion
53. ch to EDITING mode 2 Drag the spot overlay to the desired location Figure 3 17 Adjusting Spot Position 3 Right click to apply the changes To adjust the diameter of a spot 1 Double click the spot which you want to reposition to switch to EDITING mode 2 Drag one of the edge handles of the overlay to increase or decrease its diameter Figure 3 18 Adjusting Spot Diameter 3 Right click to apply the changes Performing Microarray Evaluations 87 Adjusting the alignment parameters To control the alignment process 1 On the Alignment panel of the Array Measurement toolbox check the appropriate options for the alignment process You can choose to adjust the position the overlay automatically Auto Positioning and or to separate measurement signals from background signals Segmentation In addition you can choose to apply the alignment process to selected areas only restrict to selection 2 Click Align on the Alignment panel of the Array Measurement toolbox to start the alignment process 3 Click Final Calculation to perform all remaining calculations 88 AIDA Microarray User s Manual Defining a New Template If a completely new evaluation is necessary you can either import an appropriate array template which you have previously defined and stored or which you select from the library of predefined templates shipped with the Microarray module or else create an array overlay from scratch using the Grid Defini
54. changes revealing control for calculating the ratios of selected data Settings Ratio Comparison Master 532 v7 Cut OFF Threshold w Bkg Deviation TES Bkg Multiple Ratio 100 Figure 3 70 Ratio Comparison Controls Master Specifies the master channel for the comparison The ratios resulting from the comparison are calculated by dividing the measurement value of the measurement channel by the measurement value of the master channel Cut Off Sets the cut off method Threshold Bkg Deviation or Bkg Multiple The cut off method defines a threshold If a spot s value is below this threshold the spot value is regarded as not detected Calculating ratios of data values within background range results in biased expression values since all measured data values exhibit a certain amount of random error due to the inherent bias of the cloning array production array processing spotting and scanning procedures Therefore values within background range oscillate around 0 Forming ratios using those biased background range values just yields results comprised entirely of biases To filter such biased ratios you can specify thresholds for the background range using the following controls Threshold specifies a constant background threshold value indicated by two axis parallel yellow lines in the scatter plot If the Normalized value of a spot is below this threshold value the spot value is regarded as not
55. chematic Note that enabling the Exclude from Schematic option results in a complete exclusion of the specified dots from any calculation mean and standard deviation calculation as well as Schematic Histogram 44 AIDA Microarray User s Manual Actual Probe Section In the message panel below the Exclude from Schematic section the name of the selected probe as stated in the Array Result Table is shown together with its label and the value for the measured data selected 1n the Data drop down menu of the Schematic Display section Actual Probe Configure Normalized ean 0 00 0 36 Figure 2 50 Actual Probe Section You can select the type of data to be displayed on the Actual Probe panel by clicking the Configure button and selecting deselecting the appropriate data types from the list in the Column Selection dialog Actual Probe Normalized E Columns Selection Show Columns Hide Columns Index Repeated nat used Good Mormalized 50 Log Normalized Mean Log Mormalized SD Ratio of Normalized Mean Delta Log Normalized Log Ratio Mean Log Ratio SD Expression nat used Marmalized Mean Actual Probe Normalized 0 24 0 13 Ratio of Hormalized 1 9 Figure 2 51 Configuring Actual Probe Data Display Using Microarray 45 Schematic Display Section The Schematic Display section 15 used to configure the display options for the Sche
56. cks Button v select all repetitions hide array overlay Figure 2 40 Selection Mode Section Overview Clicking on the Select Fields button activates the selection of the fields on the first array level Select Blocks activates the selection of the blocks and Select Spots Level is used to select the spots in the array Check the select all repetitions option if you want to enable the joint selection of a spot and its related duplicated spot To hide the array overlay check the hide array overlay option Settings Section The Settings section provides you with a menu for choosing various array parameter settings Settings s Alignment Background Rating Mormalisation Ratio Comparison Figure 2 41 Settings Menu For a detailed description of the parameter settings see the Setting Parameters section 38 AIDA Microarray User s Manual Array Measurement Toolbox The Array Measurement toolbox provides you with all tools necessary to evaluate arrays Array Measurement Template user defined Alignment amp Calculation Selection Mode select repetitions hide array overlay Alignment Perform v Auto Positioning Segmentation Alignment Section restrict to selection Align 3eset Template Section Selection Mode Section Rating Dot Values All Channels v Ea 4x Error Code m 1 i Rating Dot Valu
57. ction of the Array Measurement toolbox provides a toolbar used to choose different array level selections Selection Made Selection Tool 1 88 8 Spots Button Select Fields Button Select Blocks Button v select all repetitions hide array overlay Figure 2 44 Selection Mode Section Overview Clicking on the Select Fields button activates the selection of the fields on the first array level Select Blocks activates the selection of the blocks in the fields and Select Spots is used to select the spots third level in the blocks or fields 40 AIDA Microarray User s Manual Check the select all repetitions option if you want to enable the joint selection of a spot and its related duplicated spot To hide the array overlay check the hide array overlay option Alignment Section With the controls in the Alignment section you can set parameters for automated alignment processes used to match the position of the defined array spots with the real position of the dots in the array This alignment process is consequently accomplished by the integration of the intensity values of each spot Alignment Perform W Auto Positioning Segmentation restrict to selection Align Reset Figure 2 45 Alignment Section The Alignment section provides you with the following controls Auto Positioning Check this option to enable the automatic positioning of the array Segmentation Check this option
58. ctively index 1a increase remove attributes cane Figure 3 29 Specifying New Field Repetition Pattern 4 To set the repetition positions click the button next to the repeated spin box and then subsequently click in the fields of the schematic array structure displayed Pattern Definition Figure 3 30 Setting Repetitions Performing Microarray Evaluations 95 5 To complete the pattern definition process enter a name for the pattern in the Name field of the Add Field Pattern dialog and click OK Mame field1 cen Figure 3 31 Entering a pattern name To remove repetition attributes from fields 1 On the Add Field Pattern dialog click the remove attributes button 2 Click in the field whose repetition attribute you want to remove El remove att Figure 3 32 Removing repetition attributes from fields To modify an existing repetition pattern 1 Click the Pattern Definition button on the Repetition Pattern Definition section 2 On the Select Field dialog select a pattern from the list displayed above the push button controls 3 Click Modify The Modify Field Pattern dialog appears allowing you to change the pattern definition 96 AIDA Microarray User s Manual E Defining Border Lines After you completed the definition settings turn to the next step of array definition namely the definition of outer border lines To defin
59. d Definition panel of the Array Definition toolbox Note Note The buttons for defining second and third array level will become active only after defining a new overall array overlay first array level 2 Specify the number of rows and columns in the Rows and Columns spin boxes respectively 3 Apply labeling as described for the definition of the first array level above 4 Define the size of the spots by setting the spot diameter using the Diameter spin box E Array Definition Grid Definition no pattern Rows columns da 5 Diameter I EH Repeat Pattern EH 1a 1h 2a 2h Cancel Figure 3 37 Setting Spot Diameter Performing Microarray Evaluations 99 The spot diameter defines the area of intensity measurement of a given spot It has the same size for a group of simultaneously defined spots Figure 3 38 Simultaneous Defined Spots Spotting Pattern The basic structure of an array frequently contains a defined pattern of spotting where structures like empty dots or guide dots are located in certain positions and the distribution of double dotted samples 15 defined Loading Predefined Spotting Pattern To load predefined spotting pattern use the Spotting Pattern drop down list on the Grid Definition panel third level of the Array Definition toolbox EI Array Definition Grid Definition no pattern patterrn oligo hyh rzpd field 1 zod field 2 rzpd
60. ding by Sort ascending by Integral Sort descending by Integral Unselect Integral Hide Integral Settings of Integral One lined Header Select Columns Figure 2 13 Sorting Table This is especially useful for finding highest intensity values and for identifying the highest absolute or relative deviations in double dotted samples for quality control The Select Unselect options of the shortcut menu are used to select unselect a column to export The column heading of selected columns are highlighted To hide a column select the Hide option from its shortcut menu You can recover hidden columns using the Select Columns option see below Using Microarray 17 To specify the decimal digits for the value output in a column choose Settings of from the shortcut menu and enter the value for the decimal digits in the dialog that appears nm Quality Mean 01 Median Ge aL pixel Sork ascending by Integral E Sort descending by Integral 316 6064 0 p 922 4 789 0 0 00 0 0 Unselect Integral 159 5 5550 Hide Integral Settings of Integral 929 5 0 0 p 0 0 One lined Header Select Columns oor Decimal digits E Cancel Figure 2 14 Specifying Decimal Digits Display Note that the behavior of the Settings of option depends on the column you right click Columns whose cell contents is usually provided via name table import that is t
61. diting the spot diameter of loaded predefined templates The Edit Array Fields button is used to define a frame for a new array overlay or to modify an existing one Depending on whether you create a new array from scratch or edit an existing array overlay the appearance of the toolbox changes The Edit Array Blocks of Selected Fields button is used to edit the second array level The second array level is defined for selected fields of the first array level If no fields are selected on the first array level this option 1s disabled Use the Edit Array Spots of Selected Blocks button to edit the third array level The third array level 15 defined for selected fields of the first or blocks of the second array level depending on the previously defined array level structure If no fields or blocks are selected this option 1s disabled To label spots click the Define Array Labeling button The Name Table Import option is intended for easy import of names and ID numbers of samples like clones sequences oligos and so on A detailed description of the various options in this section of the toolbox is provided in the Defining a New Template section of this manual Using Microarray 37 Selection Mode Section The Selection Mode section of the Array Definition toolbox provides a toolbar used to choose different array level selections Selection Made Selection Tool E Select Spots Button Select Fields Button Select Blo
62. e 2 Wn E m X E Wn E m X E H Schematic Display c5 1235455788101234 1254567 8901134 3455783901234 Zn ruutr E n6 din run B tH t t ttt ok ook ob oo Robo EH ok EH ttt RM ot t tit FH FH tHe R a PU fb ok oM fe EU EE Ot 6 Il ROIG oe TR OE tO EF UA Ot Ot tt EE Oe A OT OR E te tt tt RE OR ER Rok oko Ue OR tO ee RS koBoRIGRRRRRA TAA eR eo OE 6 GR tt t Ax RMR e e ted t tt tt te m ttt tH ot ot HH tle tH tht GR tt FH G RM tte HH BRI tb UR RR OE Ce oRIM e Rx B IE GBRG MM m i t et tt eR t tet OF PR tb RU UR RE OP OR GB a a IM eo Oe PS PS RR tO RR C E 4oBuMoRoBokoBoRLGR
63. e Probe Table 1 Choose View Probe Table 20 AIDA Microarray User s Manual Data Description The possible columns of the Probe Table are the following Table 2 2 Probe Table Columns Column Short Description Index Running table row number Name Name of the probe user definable corresponds to the Name column of the Array Result Table ID ID of the probe user definable corresponds to the ID column of the Array Result Table not used 1 User definable columns corresponds to the respective columns of the Array Result Table not used 2 User definable columns corresponds to the respective columns of the Array Result Table Repeated Number of repetitions of the probe in the array as determined by the spotting pattern Values listed for each channel mean and standard deviation values are calculated using the good spot values of a channel from the result table of a probe Good Number of repetitions not flagged for Bad quality Normalized Mean Mean of the Normalized values Normalized SD Standard deviation of the Normalized values Log Normalized Mean Mean of the Log Normalized values Log Normalized SD Standard deviation of the Log Normalized values Ratio of Normalized Mean Ratio of the Normalized Mean values with Ratio Comparison Settings applied Expression Ratio of Normalized Mean encoded according to the following scheme On value of master within background range and client s value abov
64. e background Off value of master above background and client s value within background range Ratio ratio value gt 1 1 Ratio ratio value lt 1 Signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set specified in the Image Attributes dialog Using Microarray 21 Table 2 2 Probe Table Columns Column Log Ratio Mean Log Ratio SD Delta Log Normalized Short Description Mean of the Log Ratio values Standard deviation of the Log Ratio values Log Normalized value of the channel minus Log Normalized value of the master channel Signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set specified in the Image Attributes dialog 22 AIDA Microarray User s Manual Array Scatter Plot In the Array Scatter Plot the Normalized or Log Normalized spot values of the measurement channels are plotted against the spot values of the master channel The values of Min Expression red and green lines Max Ratio yellow lines and Bkg Cut Off yellow colored Bkg dots configurable via the Ratio Comparison panel of the Array Definition toolbox are represented O x Channel 635 w 1 Data Normalized 5 1 Normalized 0 0001 0 01 0 1 1 532 7 Normalized Figure 2 19 Array Scatter Plot Example To open the Array Scatter Plot window e Choose
65. e the outer border lines of the array overlay 1 Set the vertical edge of the array by clicking two positions in the Image window Figure 3 33 Setting Vertical Edge 2 Then move the mouse pointer to stretch the frame Figure 3 34 Stretching the Frame Performing Microarray Evaluations 97 Ea esol a i ei la Figure 3 35 Frame Definition Accomplished The new array overlay changes its status from EDIT red to ACTIVATED magenta Defining a Block Matrix Field If necessary you can define a block matrix which further divides the sub areas determined by the overall array definition field matrix definition see above into smaller sub areas On the Grid Definition panel of the Array Definition toolbox click the Edit Array Blocks of selected fields button to edit the block matrix of selected fields Figure 3 36 Defining Block Matrix Again the Grid Definition panel changes its appearance The options for specifying columns rows spacing labels and repetition pattern provided by the control panel are identical to the ones used to define the overall array see section above 98 AIDA Microarray User s Manual Defining a Spot Matrix Block The spots form the basic structure of an array and are given in rows by columns To specify the spot matrix which determines the number of final spots 1 Click the Edit Array Spots of selected blocks button on the Gri
66. ead of this the set of not regulated genes is given by the set of reference dots For all channels 2 1 d in the array let hj spot arcsinh a b Signal spot 20 denote the variance stabilization transformation proposed in Huber et al 2002 which is calculated from the set of reference dots as not regulated genes Huber W von Heydebreck A S ltmann Poustka A Vingron M 2002 Variance stabilization applied to microarra data calibration and the quantification of differential expres sion Bioinformatics 18 Suppl 1 96 104 138 AIDA Microarray User s Manual Than LogNormalized spot h spot 21 Normalized spot 2 pot 22 Re ference spot not defined 23 Normalization by Data Range Let Signal denote the measurement value of a spot which is used for the normal ization Let D d i 1 N denote the set of applied measurement dots within the entire array After sorting D by signal values percentages of the amount N are rejected from the small and the large values side respectively This leads to the set D 4 i 1 N of dots which are used for calculating a global normalization factor for the array channel Arithmetic Mean 1 7 C D Signal d 24 Reference spot C 25 Normalized spot ES 26 Geometric Mean C exp 5 log Signal 9 27 Reference spot C 28 Normalized spot _ 29 Appendix 139
67. ean or the median value should be used for background calculation Background spots can be marked by color or label 108 AIDA Microarray User s Manual Global Background Dots The Mean or the Median value of all selected spots flagged as Bkg or 15 averaged and the result is used for background subtraction Consequently the same background value is subtracted from each spot intensity Global Bkg Dots use empty dots Bydot Meant Median Background Dot Selection Tool Remove Background Flag Button Background Dot Deselection Tool Assign Background Flag Button Markby Color Label Figure 3 51 Controls in Global Background Dots Mode To mark a spot for background subtraction 1 Choose Background from the menu in the Settings section of the Array Definition toolbox 2 Choose Global Bkg Dots from the menu directly below the Settings menu 3 Choose the Selection tool from the Selection Mode panel of the Array Definition toolbox 4 Click the designated spot 5 Click the Assign Background Flag button in the Settings section Selection Made 88 222 E aoa v select all repetition hide array overlay use empty dots Ey dot Meant Median Markby Color Label Figure 3 52 Assigning Background Flags via Assign Background Flag Button Performing Microarray Evaluations 109 Alternative procedure 1 Choose Background from the menu in the Settings section o
68. er to properly insert the rows of the source columns into matching row slots of the target table Array Result Table the position of the particular label column in the source table import table must be specified Microarray provides four methods for associating table rows 1 Import by Global Label 2 Import by Hierarchic Label 3 Import by Global Position 4 Import by Hierarchic Positions The Import by Global Label methods associates rows using the Label column Import by Hierarchic Label uses the Field label Block label Spot label columns Import by Global Position utilizes the Row Index and Column Index columns whereas the Import by Hierarchic Positions method associates source and target rows via the Field Row Block Row Spot Row and Field Column Block Column Spot Column columns To import Name Tables 1 Perform one of the following tasks Click the Name Table Import button in the Grid Definition section of the Array Definition toolbox Click the Import menu item on the File menu and then choose Array Results from the cascading menu The Name Table Import dialog appears 128 AIDA Microarray User s Manual raytest 2 Select the file you want to import from the list of available files and click Open The file has to be in the Array Name Table or Table ASCII File format EH Array Definitian Name Table Import nco c adf alx Grid Definition Look inc Examples 4m t E ejm File name Jo
69. es Section Figure 2 42 Array Measurement Toolbox Overview To display the Array Measurement toolbox Choose Evaluation gt Array Measurement Alternatively you can select the Array Measurement button on the toolbar Using Microarray 39 Template Section The Template section of the Array Measurement toolbox 15 used to import predefined array templates Template onco atf 1 22 03 HL Import Array Template Button Status Panel Name Table Import Button Alignment amp Calculation Figure 2 43 Template Section Overview To import a predefined array template from the template library shipped with AIDA Microarray click the Import Array Template button The Name Table Import option is intended for easy import of names and ID numbers of samples like clones sequences oligos and so on The status panel shows the name of the currently applied imported template The Alignment amp Calculation button is used to start the automated alignment and calculation measurement background calculation rating normalization process The Alignment and Calculation process is applied to the entire array regardless of the current selection and restrict to selection settings For a detailed description of the various options provided by this section of the toolbox see the Defining a New Template section of this manual Selection Mode Section As with the corresponding section of the Array Definition toolbox the Selection Mode se
70. ettings panel 2 Select a spot in the Image window To remove a previously assigned reference flag from a spot 1 Click the spot s you want the reference flag to be removed 2 Click the Unref button on the Settings panel Alternative procedure 1 Click the Reference Dot Deselection tool on the Settings panel 2 Select the spot in the Image window you want the reference flag to be removed Reference spots can by marked by color or label Just specify the marking by selecting one of the Mark by radio buttons Performing Microarray Evaluations 121 Data Range Normalization Method Data Range represents a normalization method where guide dots empty dots and dots with low high signal intensity can be excluded from referencing Settings Mormalisation Data Integral Method Data Range Ignore Upper 25 0 Ignore Lower 25 0 exclude empty dots exclude guide dots exclude bad dots Figure 3 69 Data Range Normalization Method Data Range is a global normalization method normalizing all spots using the same reference value Each normalization result of a spot is referred to either by the Arithmetic Mean or the Geometric Mean of the reference spot See the Formal Description of Normalization section in the Appendix for details 122 AIDA Microarray User s Manual Ratio Comparison Settings If you choose the Ratio Comparison menu item the appearance of the lower part of the toolbox
71. f the Array Definition toolbox 2 Choose Global Bkg Dots from the menu directly below the Settings menu 3 Activate the background definition mode by clicking the Background Dot Selection tool on the Settings panel 4 Then select a spot in the Image window use empty dots dot Meant Median sss 5 aw Color Lab Figure 3 53 Assigning Background Flag via Background Dot Selection Tool In addition you can specify whether the background spots are marked by color or by label using the respective controls on the Background Definition panel To remove a previously assigned background flag from a spot 1 Click the spot s you want the background flag to be removed 2 Click the Remove Background Flag button on the Settings panel Alternative procedure 1 Click the Background Dot Deselection tool on the Settings panel 110 AIDA Microarray User s Manual 2 Then select the spot in the Image window you want the background flag to be removed use empty dots dot Meant Median Markby Color Lab Figure 3 54 Removing Background Flag via Background Dot Deselection Tool If the use empty dots option is enabled the spots which are defined as empty in the spotting pattern are used for background subtraction Weighted Background Dots For selecting deselecting weighted background dots the same selection methods are available as for the selection of global backgro
72. f the array AIDA Microarray directly imports the information of the GAL template file and uses this information to automatically build its internal array geometry and name table To import a GAL template 1 On the File menu click the Import menu item and then select Spotter Template from the cascading menu The Spotter Template Import dialog appears Spotter Template Import Michelle1 E Look in E GAL example aa Tu amp Chip Axon gal Chip Axon patch gal File name Files af type GenePis Array List Cancel Figure 2 79 Spotter Template Import Dialog 2 Select a GAL file and click Open T2 AIDA Microarray User s Manual Figure 2 80 Array Overlay And Nametable Created From Imported Data Importing Name Tables To import nametables names and ID numbers of samples to the Array Result Table use the Import option of the File menu or alternatively the Name Table Import option of the Array Definition or Array Measurement toolbox The Name Table Import dialog appears allowing you to specify the options for the name table import The table has to contain the same labeling as defined in the array for details see the Import of Name Tables section below Using Microarray 73 raytest 74 AIDA Microarray User s Manual Performing Microarray Evaluations Standard Evaluation Procedure With a few simple steps precise data from array experiments can be generated usin
73. f the column whose width you want to change 2 Drag the border to the appropriate direction 3 Release the mouse button FT Array Result Table Onco_c adf Indes Label Integral b 5 11 D03An2h 3h t Array Result Table Onco_c adf Indes Label Integral L PSL 11 D03An2h 3h 2 Hi Array Result Table Onco_c adf 3 Index Label Type Integral Bkg 4 PSL PSL 5 11055025 3b 28 4 14 8 Figure 2 11 Changing Column Width Shortcut Menu of the Array Result Table Each column in the Array Result Table has a shortcut menu attached Hi Array Result Table c raytest Aida Image Analyzer dev 4 00 022 Examp es m x Area Integral W Quality Median GL Bk GL pixel 1 Sort ascending by Integral j 1 j EE Sort descending by Integral 316 60640 B22 4 789 0 0 00 E Unselect Integral 0 0 318 6050 0 8 1734 6 159 5 355 0 316 2998 0 Settings of Integral 954 2 134 5 829 5 1 315 910 4 127 0 321 5 316 5851 0 6072 0 NEN 461 6 1158 5 1399 0 873 5 54 5 9995 316 99220 5 One lined Header 841 1 796 0 000 000 0 00 316 958 0 09400 5442 1300 4 1 Figure 2 12 Shortcut Menu Array Result Table 16 AIDA Microarray User s Manual The table can be sorted by any value column in ascending order A to Z or zero to 9 or descending order Z to A or 9 to zero Just choose the appropriate menu item Sort ascending by Sort descen
74. g the automatic evaluation feature provided by AIDA Microarray The steps of the standard evaluation procedure described below requires that an suitable array template already exists This can be either an previously saved user defined template or one of the built in pre defined templates shipped with AIDA Microarray If there is no suitable template available you either have to define a new one from scratch see the Defining a New Template section for details or to modify an existing template see the Manual Alignment Adjustments section for details Step 1 Loading the Image File To load a multi channel image file FUJI SET file or multi channel TIFF file 1 Choose File gt Open 2 Select the image you want to load from the AIDA Data dialog and click Open 75 3 In the Open Multi Channel Data Set dialog check the channels to be loaded im Open Multi Channel Data Set E E _ A DataSet c sPragram Filessraptests amp ida Image Analyzer dev 4 C 2 3 E amples sE samples Microarray sE sample 1 47 22 PZ Excitation Emission Color Emission Color zl Excitation Channel 1 Excitation Emission Color 635 rT 535 red Channel 2 Excitation Emission Color 32 2 532 green Open Cancel Figure 3 1 Open Multi Channel Data Set Dialog 4 Choose the display color for each channel from the respective Color menus 5
75. hannels are plotted against the spot data of the master channel The values of Min Expression red and green lines Max Ratio yellow lines and Bkg Cut Off yellow colored Bkg dots configurable via the Ratio Comparison panel of the Array Definition toolbox are represented The mean of the Log ratios of the data sets gray line and the data mean thresholds color coded lines configurable via the Array Schematic toolbox are represented Probe Scatter Plot c raytest 4ida Image Analyzer dev 4 00 023 Exa z mi gt Channel 635 w 1 Data Normalized 1 qi qi Bo 01 m 001 Wa 0 0014 0 0001 0 001 0 01 0 1 1 532 7 Normalized Mean Figure 2 28 Probe Scatter Plot Example Using Microarray 27 To open the Probe Scatter Plot e Choose View gt Probe Scatter Plot Scatter Plotwindow so please refer to the respective description in the Array Scatter Plot section for further details The Schematic Display window provides you with a schematic drawing of the array In this data view the mesurement values of the spots of a channel are color coded Any value of any channel can be selected using the Schematic Display panel of the Array Schematic toolbox All data within a specific data range are represented by spots of The Probe Scatter Plot window provides the same set of display controls as the Array identical color Schematic Display Window TNT o
76. he Name and the ID column as well as the two user definable columns with the default name lt unused gt have Column Settings dialog boxes totally different from the ones provided by measurement value output columns Column Settings Name eoo Jer Enter here the whole URL link to an internal or external database Place a 61 atthe position where the cell value is ta be expanded in Cancel the URL query Figure 2 15 Column Settings Dialog Name Column Instead of decimal digit specifications you can edit the column s default name and enter a URL Uniform Resource Locator to link the column s cells to internal or external databases see the Importing Name Tables section for details Use the One lined Header Two lined Header option to reduce or expand the column heading by one line the second line indicates the units of measurement 18 AIDA Microarray User s Manual To select the columns that should be displayed in the table choose the Select Columns option w Sort ascending by Integral Bkg Sort descending by Integral Bkg Select Integral Bkg Hide Integral Bkg Settings of Integral Bkg One lined Header Figure 2 16 Select Columns Command of the Shortcut Menu A dialog appears displaying two lists titled Show Columns Hide Columns Select the columns you want to show or hide and click the appropriate arrow symbol to transfer the items between the two lists To unselect a particular ite
77. ing and Y Spacing spin boxes respectively The defined sub areas will be separated by the distances specified Performing Microarray Evaluations 91 4 Click the downward arrow in the left corner of the Labeling section directly below the X Spacing and Y Spacing spin boxes to determine whether to apply labels to the sub areas of the array and how the labeling is performed A drop down menu appears allowing you to choose between the labeling types No Labels Rows by Columns Columns by Rows Sequential and Repeat Pattern label wv Rows by Columns Columns by Rows Sequential Repeat Pattern Figure 3 23 Labeling Type Drop Down Menu 5 To define labels for rows and or columns click on the respective heading of the Labeling section 6 Pick the appropriate label by numbers uppercase or lowercase letters for your columns and or rows from the drop down menu that opens 001 002 Essa Set Prefix 001 002 Set Prefix Figure 3 24 Selecting Labels for Rows and Columns 92 AIDA Microarray User s Manual Cancel In addition you can set a prefix Set Prefix option to separate the rows and columns by a special character or to separate this label level from the labels of the upper array level in order to avoid confusion in the connected label for spots 1f you are using hierarchical labels for the spots yos
78. ing the respective spin box After performing the necessary modification to the overlay click OK or alternatively right click on the overlay to confirm the changes and switch the status of the overlay to ACTIVATED Performing Microarray Evaluations 79 The message box to the left of the Import Array Template button shows the name of the imported template Ren Array Measurement Template Clontech Atlas Large Array E Alignment amp Calculation Figure 3 6 Template Import Completed Step 4 Starting Automatic Alignment and Calculation To start the automatic alignment and calculation process 3 On the Array Measurement tool click Alignment amp Calculation Ren Array Measurement Template at 8 2 2 03 Deale ea REA Figure 3 7 Starting Automatic Alignment and Calculation Process The alignment process is performed in two steps the positioning of the overlay and the separation of the measurement signal from the background signal so called segmentation Alignment amp Calculation performs the steps in sequence Positioning and segmentation of the spots is done for all channel simultanously For internal processing purpose the values of each channel are weighted using the standard deviation of the intensity values of the background pixels This allows the comparion of the channel data During the positioning process the blocks of the array are aligned according to the maximum intensity val
79. l Array Analysis Table Clauses x Alternatives Clauses 1 Clause list Add Delete Delete OK Cancel Figure 2 58 Adding rule clauses This opens the Analysis Table Clauses dialog which allows you to define your rule expression Analysis Table Clauses m 8 Analysis Table Clauses E X C Ems Analysis Table Clauses _ Integral PSL 60 0 Figure 2 59 Specifying Rule Expression 50 AIDA Microarray User s Manual Publishing Layout Toolbox in Microarray In Microarray the standard AIDA Protocol Print toolbar of the Publishing Layout toolbox is expanded by four additional buttons for specifying the print options for the Probe Table Array Scatter Plot Probe Scatter Plot Schematic Display Schematic Histogram Repetition Deviation and the Array Analysis Table Publishing Layout Annotate fal Protocol Print Print Preview b Page Layout Edges Jas 20cm Bottom 20cm Lett zom Right 20cm Page Layout Button Image Button Probe Table Button Array Scatter Plot Button Probe Scatter Plot Button Schematic Display Button Header Iv File Path Page Number Current Date Schematic Histogram Button Repetition Deviation Button Footnote Author Last Change Time v Software Version Analysis Table Button Figure 2 60 Publishing Layout Toolb
80. labels of each level to the label of the spot Hi Array Result Table nco x Figure 3 43 Hierarchic Labeling Example 1 The hierarchic labels shown in the screenshot example above are composed of two letter number identifiers representing the two levels of the array The first identifier A1 denotes the block in the first row A of the first column 1 of the field matrix the second identifier a2 denotes the spot third level in the second row 2 of the first column a of block A1 field matrix The settings for this example are the following Table 3 1 Settings for Hierarchic Labeling Example 1 Columns Rows Labeling Type Label Type Rows Columns Field Matrix 16 24 Rows by Columns uppercase letter number Block Matrix n a n a n a n a n a Spot Matrix 5 5 Rows by Columns lowercase letter number with prefix Performing Microarray Evaluations 103 In addition it is also possible to label spots using the numbers defined in repetition patterns This allows you for example to identify corresponding clones from the same microtitre plate To enable this type of spot labeling select the Repeat Pattern option from the labeling type drop down menu Array Definition Grid Definition E oligo hyh pu inh Diameter 1 00mm Repeat Pattern EM label Rows by Columns Columns by Rows Sequentia w Repeat Pattern
81. lays Grayscales Scales and Overexposed can be enabled by checking the respective options The printout can be configured to automatically fit to the page or the relative size of the image representation can be selected in In the Analysis Table Options and Probe Table Options sections you can specify to print the entire table full table or just a part of it rows above as well as the paper orientation landscape or portrait The paper orientation can also be specified for the Schematic Display output using the drop down menu in the Schematic Display Options section 60 AIDA Microarray User s Manual Preview As for printing in AIDA there are two ways of previewing pages before they are actually printed 1 Using the Print Preview option of the File menu or 2 using the Preview button on the Publishing Layout toolbox The Print Preview option of the File menu opens the Preview dialog which allows you to specify the layout settings for the preview The options presented here are the same as in the Print Microarray dialog and those selectable from the various sections of the Publishing Layout toolbox However they are not stored with the data file The Preview option of the Publishing Layout toolbox allows you to preview all pages as configured in the Publishing Layout toolbox before they are actually printed Clicking the Preview button opens the Preview window directly since all layout setting are already made through the Prot
82. lbox you can configure the settings for the schematic analysis of the array 85 Array Schematic Exclude from Schematic Empty Dots F Guide Dots Background Dots Exclude From Schematic Section Reference Dots Bad Quality Dots Actual Probe Configure Actual Probe Section Schematic Display Data i Channel 532 WW Mean 50 Colors 34 SDPich 2 0 4 20 380083 Schematic Display Section 16 876 8 2 0 4 255 4 asl Figure 2 47 Array Schematic Toolbox Overview Using Microarray 43 Exclude From Schematic Section In the Exclude from Schematic section of the Array Schematic toolbar you can select some dot types for exclusion from the schematic analysis of the array These types are Empty Dots Guide Dots Background Dots Reference Dots and Bad Quality Dots Exclude from Schematic Empty Dots Guide Dots Background Bots Reference Dots Bad Quality Dots Figure 2 48 Exclude From Schematic Section Values excluded via the Exclude from Schematic option appear in gray in the Schematic Display window and are marked with a cross to avoid confusion with real gray scale values gt H Schematic Display Onco_c adf E 01 02 03 00000 60 66 00 oo eoe o0 o0 00 00 060 A eoe o0 00 oe 0 xXOOO eoe 00 S gt 2a eoe 00 00000 00000 00000 00 00 00 00 00 oo eo 00 00 00 00 oo Figure 2 49 Dots Excluded From S
83. lignment the program moves each dot above the threshold to the nearest maximum of averaged intensities The dots below this threshold or dots where no maximum is found are set by the program to a position which is calculated from the aligned dot positions Figure 3 8 Aligned Array Example Spots with at least one overexposed pixel magenta are automatically marked as bad and tagged with the label sat for saturated in the Quality column of the result tables Performing Microarray Evaluations 81 Step 5 Saving the Evaluation After the Alignment amp Calculation process is completed save the evaluation with the automatic export option enabled All measurement data are exported to corresponding files To save the evaluation 1 Choose File gt Save or File gt Save New Evaluation As 2 In the Save New Evaluation As dialog select a location enter a file name and click Save Manual Alignment Adjustments In most cases the automatic alignment process described in the previous section successfully aligns defined array spots and real dots But sometimes it fails due to irregular dotting patterns In this case you might want to adjust the alignment manually You can do this by using the standard overlay editing features provided by AIDA Microarray You can perform the following three modification Adjusting parts of the overlay for better fitting with the image Adjusting the position of particular dots
84. ller Seco 6 44 45 Se d 49 Publishing Layout Toolbox in 51 Print Options for Probe 52 Print Options for Array Scatter 1 53 Print Options for Probe Scatter 1 54 Print Options for Schematic 1 1 55 Print Options for Schematic Histogram 56 Print Options for Repetition 57 Print Options for Analysis 58 lure RI TC 59 Pie leues bee ie PRISE ILE E CELO DU Ae epu Ea ds 61 AIDA Microarray User s Manual Export aud yer pas Pd EDS S RUNI 62 Exporting a ege Eae phon Eta ed onda abun s 62 Exporting Array Result 62 Exporting Probe Tables esta eed aces ined ee de 63 Exporting Array Scatter 64 Exporting Probe Scatter 1 65 Exporting Schematic 1 65 Exporting Schematic 1 65 Exporting Analysis Table isis oc Ex RIA RIO REA AEQ A E 66 Automatic Export of
85. log You can specify the Range of the exported rows and columns as well as the List Delimiter and Decimal Delimiter in the respective panels of the dialog 4 Complete the action by clicking the OK button The data can be saved to a simple ASCII text or MS Excel file Exporting Array Scatter Plots To export Array Scatter Plots 1 Choose File gt Export gt Array Scatter Plot 2 In the Array Scatter Plot Export dialog that appears enter a name for the export file 3 Complete the export action by clicking the Save button The data are saved to a file in the Windows Metafile or Windows Enhanced Metafile format depending on your file type selection 64 AIDA Microarray User s Manual Exporting Probe Scatter Plots To export Probe Scatter Plots 1 Choose File gt Export gt Probe Scatter Plot 2 In the Probe Scatter Plot Export dialog that appears enter a name for the export file 3 Complete the export action by clicking the Save button The data are saved to a file in the Windows Metafile or Windows Enhanced Metafile format depending on your file type selection Exporting Schematic Display To export the Schematic Display 1 Choose File gt Export gt Schematic Display 2 In the Schematic Display Export dialog that appears enter a name for the export file 3 Complete the export action by clicking the Save button The data are saved to a file in the Windows Metafile or Windows Enhanced Metafile format dependi
86. lumn in the Table Header Name text box 8 Enter the query string for database search using database specific query syntax in the Link text box Alternatively you can select predefined previously defined query strings from a list a Click the button next to the Link text box The Table URL Links dialog opens b Choose an entry from the list box and click OK aene Bank Figure 3 82 Selecting Query String From the Table URL Links Dialog Performing Microarray Evaluations 133 134 AIDA Microarray User s Manual Appendix Formal Description of Background Subtraction Methods Global Grid Background Dots Let 16 i 1 N denote the set of all background spots in the array or in a block background spot is defined as spot which either has the background attribute set or is marked as empty use empty dots option checked Let Signal denote the Mean or Median value SD the standard deviation of the pixel intensity values for a spot The background for all spot in the array or block is calculated as follows X Signal b Area Area bj 3 SD bi Area b Area bj Bkg BkgS D Weighted Background Dots Let Signal denote the Mean or Median value and SD the standard deviation of the pixel intensity values for a spot Let b 1 N denote the set of all background spots in the array background spot is defined as spot which either
87. m just click it again Columns Selection 2 X Show Columns ennt Row Index Column Index anat used anat used Integral Bkg GL Median Bkg GL Field Row Field Column Black Row Black Column Mean Bkug AL Spot Raw Integral SD Spot column Akg SD Field Row Label Field Column Label cancel v Position pixel Diameter pixel Area pixel Figure 2 17 Columns Selection Dialog Using Microarray 19 Probe Table The Probe Table lists the averaged values of the repeated spots of a probe Only good repetitions are used for the calculation of the averaged values Values of multiple channels are listed in numbered child columns of a parent column Hi Probe Table c Program Files rayEbesb Aida Image Analyzer dev 4 00 02 ER mi xj Index Repeated Name ID enot used 8009 Normalized Mean Normalized SD Log N 1 2 1 2 1 2 1 1 15 15 15 0 14 0 10 0 0 0 0 5 ep 140 02 02 200 8 L4 c9 deep ow s Ls je oc o ox 02 ef ve se om or Lp 9 17 o ate 145 02 02 ate free 207 oe sof ve jeep 226 170 02 Cp 9 or on Pape raha sar ow 02 02 Pale eps 00 200 02 02 Pape frets c00 oo 00 asf ve free 0 15 15 15115 1 12 0 83 0 1 0 1 4 k Figure 2 18 Probe Table Example B To display th
88. ma Setting for Report Columns Index Included in Export Figure 2 75 Edit Automatic Export Setting Dialog The dialog provides you with the following option Export on Save Automatic enables the automatic export and generates the export filename automatically from the evaluation filename No disables the automatic export If you select Prompt for you will be prompted for an export data filename when you save an evaluation Parameter enables disables the export of the table s parameter pane Table Header enables disables the export of the table header List Delimiter allows you to select a delimiter for the columns Decimal Delimiter allows you to select a decimal delimiter format Settings for Report Columns select columns subsequently and click to select or deselect the Included in Export option to include exclude the respective column Depending on the column you selected you can specify additional settings in this example the Decimal Digits for the exported values 3 Select the appropriate options and click OK to complete the action Using Microarray 69 Saving Automatic Export Settings 1 On the Automatic Export Setting dialog click Save Setting The Save Automatic Export Setting dialog opens Im Save Automatic Export Setting 2004 04 29 adF Mame example ID 2004 04 29 Cancel Figure 2 76 Save Automatic Export Setting Dialog 2 Type a unique name and an ID for
89. matic Display the Schematic Histogram and the Repetition Deviation window schematic Display Data Mean Bkg d Data Drop Down Menu Channel 532 v7 Channel Selection Menu Colors Spin Box Color Coding Drop Down Menu Miniaturized Schematic Histogram 31323 Data Range Spin Boxes 15595 68 aL Figure 2 52 Schematic Display Section Overview Select the measurement value and channel to be displayed in the Schematic Display window from the Data and Channel drop down menu respectively To configure the color output of the Schematic Display window choose the number of colors 3 5 or 7 you want the data to be reduced to using the Colors spin box Specify the color coding scheme for the data via the drop down menu displayed to the left of the Colors spin box Either the data of interest within a specified range are displayed in one of the colors Data X option or the deviation to the mean value is coded by the colors Mean SD option If the Data X option is selected set the data range for each color by dragging the border of the respective color bar in the miniaturized Schematic Histogram with the mouse or by entering the explicit values If the Mean SD option is selected this 46 AIDA Microarray User s Manual feature is disabled Instead the deviation of the spots to the mean value of the data of interest is given in units of 10 standard deviation SD Enter the step size in the SD Pit
90. ment process is consequently accomplished by the integration of the intensity values of each spot The alignment process can be varied by the following parameters Local Positioning Adjusts the position of the array to the image locally Segmentation Choose Fixed Diameter to enter a fixed diameter value for all dots or choose Adapted Diameter to let AIDA determine the diameter of each dot automatically If you choose Adapted Diameter you must specify the allowed diameter variation range using the Dia max Dia min spin boxes Performing Microarray Evaluations 107 Background Settings If you choose the Background menu item the appearance of the lower part of the toolbox changes revealing control for setting background parameters settings Settings Background Local Dot Ring Background Inflate Dots O01 0cm Global Bkg Dots Ring Width 01cm Weighted Bkg Dots Grid Dots area Mean Median Lowest Grid Dots Local Dot Ring Local Grid Ring Mode of Non Spot Global Image Regions Weighted Image Regions Figure 3 50 Background Controls The intensity values of all spots which have been aligned and integrated can be corrected for background Nine different background subtraction methods can be selected from the Background menu in the Settings section of the Array Definition toolbox For all these background subtraction methods you can specify if the m
91. mns are only available if a comparison with a master channel was performed Ratio Ratio of the normalized values with Ratio Cut Off values applied Log Ratio Binary logarithm of the Ratio value Expression Value of the Ratio column encoded according to the following scheme On result value of master within background range and client s result value above background Off result value of master above background and client s result value within background range Ratio ratio value gt 1 1 Ratio ratio value lt 1 Signal unit and length unit units of measurement depending on the type of the imaging device used to obtain the image and the options set specified in the Image Attributes dialog You can select the number and type of columns you want to display using the shortcut menu of any column in the window see below To change the column order 1 Click the heading of the column to be moved hold down the mouse button and drag to the desired position in the table 2 Release the mouse button t Array Result Table Onco_c adf Index Label Tuna A Wi Array Result Table Onco_c adf ES zm e a array Result 1 Label 02402b i B A 2e 4jUA02b T 2 068025 fi 8104405 5jAUe 8 A03e 34035 81044025 Figure 2 10 Changing the Column Order Using Microarray 15 To change the column width 1 Point to the border o
92. n m Calculation Method Menu exclude bad dots Reference Dot Selection Tool __ fet Unref Button Ref Dot Deselection Tool Ref Button eference Dot Deselection Too Dese HET Figure 3 68 Reference Dots Controls 120 AIDA Microarray User s Manual There are three normalization domains available which can be selected using the Area menu Entire Array Array Field and Array Block If you choose Entire Array the reference value results from averaging the values of all reference spots in the entire array Array Field averages the values of all reference spots in an array field whereas Array Block averages the values of all reference spots in an array block Each normalization result of a spot is referred to either by the Arithmetic Mean Geometric Mean or the Variance Stabilisation value of all spots or to individually selected normalization spots Normalization 15 done using the same reference value for all spots See the Formal Description of Normalization section in the Appendix for details Bad dots can be excluded from normalization by checking the exclude bad dots option To mark a reference spot normalization spot 1 Choose the Selection tool from the Selection Mode panel of the Array Definition toolbox 2 Click the designated spot 3 Click the Ref button on the Settings panel Alternative procedure 1 Activate the reference dot definition mode by clicking the Reference Dot Selection tool on the S
93. nco tet Files of type Mame T able Cancel Figure 3 74 Selecting Name Table File for Import The Import Name Table dialog appears allowing you to specify the options for the name table import via spin boxes and tabs In the lower part of the dialog the import source table is displayed Import Name Table Clontech Large Array adf E X Link Link 2 Link 3 Link 4 Import by Global Label Column 2 Table Header Mame Mame Link Delimiter 1 Gene Mame GenBank SwissProt Ac Atlas Human c myc oncage VOO566 PO1106 Ala macrophage c 03663 0 333 Alb myb proto or M15024 10242 Alc 06182 210721 Ald 07594 103162 13293 10244 Alf 24888 P20393 Alg 212794 210588 cancel Figure 3 75 Import Name Table Dialog Performing Microarray Evaluations 129 3 Select the type of delimiter TAB comma semicolon or SPACE from the Delimiter drop down list 4 Specify the import method using the pull down menu below the Delimiter drop down list Import Name Table nco c adf Delimiter lt gt Lir T Cc w Import by Global Label Import by Global Position Import by Hierachic Label Import by Hierarchic Positions Lir zl Figure 3 76 Selecting Import Method 5 Specify the associations between source table the name table to be imported and target table the actual array result table depending on the import method you selected If
94. ng on your file type selection Exporting Schematic Histogram To export the Schematic Histogram 1 Choose File gt Export gt Schematic Histogram 2 In the Schematic Histogram Export dialog that appears enter a name for the export file 3 Complete the export action by clicking the Save button The data are saved to a file in the Windows Metafile or Windows Enhanced Metafile format depending on your file type selection Using Microarray 65 Exporting Analysis Table To export the Analysis Table 1 2 4 66 Choose File gt Export gt Analysis Table In the Analysis Table Export dialog that appears enter a name for the export file Click Save to confirm the export The Analysis Table Export Options dialog appears allowing you to set further option for the export of the analysis table Analysis Table Export Options E X Table Header Cancel i All Rows Selected Rows Cancel All Columns Selected Columns List Delimiter Decimal Delimiter Tabulator C Point Comma System Detaults Figure 2 73 Analysis Table Export Options Dialog C Comma C Semicolon You can specify the Range of the exported rows and columns as well as the List Delimiter and Decimal Delimiter in the respective panels of the dialog Complete the action by clicking the OK button The data can be saved to a simple ASCII text or MS Excel file AIDA Microarray User s Manual Automatic Export of
95. ng the appropriate radio button in the Format section The Graphic option in the Print section enables or disables the print output of the graphic displayed in the Schematic Histogram window Protocal Print Print Preview Schematic Histogram Format Portrait Landscape L Graphic Figure 2 65 Schematic Histogram Print Options 56 AIDA Microarray User s Manual Print Options for Repetition Deviation The Repetition Deviation button on the Protocol Print toolbar allows you to configure the layout setting for the Repetition Deviation window You can select the paper orientation Portrait or Landscape by clicking the appropriate radio button in the Format section The Graphic option in the Print section enables or disables the print output of the graphic displayed in the Repetition Deviation window Protocal Print Print Preview Repetition Deviation Format Fortrait Landscape Graphic Figure 2 66 Repetition Deviation Print Options Using Microarray 57 Print Options for Analysis Table Clicking the Analysis Table button switches to the layout setting controls for the Analysis Table window To select the paper orientation Portrait or Landscape click the appropriate radio button in the Format section In the Print section the scope for the print output can be specified To enable printing of the table check the Table box and specify the appropriate output e
96. ocol Print panel 8 Aida Microarray Preview 2004 04 29 adf BENED 18 File Edit Evaluation Options View Window Help zm BEE aja Close 4 e Page 1 of 153 Moca Sakma Mey 01 04 1 53 41 C ABamplesiBemples 2114 0 29 201 E LLEI Aahar Sys iem Adminis rake L Charged Thrsday Apri 29 2004 6 47 D Alda is Version 00023 Figure 2 70 Preview Example Using Microarray 61 Export and Import In AIDA Microarray array definition as well as the content of all windows can be exported to a specific file Exporting Templates You can save your own array definition including all evaluation parameters for example background definition parameters reference definition parameters column selection to a template file and import it to any other image To export your own array definition 1 Choose File gt Export gt Template 2 In the Export Template dialog that appears enter a name for the export file and confirm the action by clicking Save Or 1 Click the Export Template button on the toolbar to open the Export Template dialog 2 Enter a name for the export file in the File name text box and complete the export action by clicking Save Exporting Array Result Tables To export the Array Result Table 1 Choose File gt Export gt Array Result Table 2 In the Array Result Table Export dialog that appears enter a name for the export file 3
97. ow Example To open the Schematic Display window Choose View Schematic Display Using the Schematic Display panel on the Array Schematic toolbox you can configure the Schematic Histogram and the Schematic Display window to display certain ranges of data values in different colors Schematic Display Data Mean Bkg E Channel 532 7 T Data Colors 7 m T Schematic Histogram c ExXamples Examples Micr J rj x 300 200 a 100 10000 20000 30000 40000 532 7 Mean Bkg GL Figure 2 32 Defining Color Coding Scheme 40311 1 33570 5 26830 0 20089 4 122489 6608 4 aL 30 AIDA Microarray User s Manual For more detailed view of the histogram the Schematic Histogram window provides zoom function similar to the one provided by the Array Scatter Plot window see the Array Scatter Plot section for a detailed description of how to use the zoom function Shortcut Menu of the Schematic Histogram Window The shortcut menu of the Schematic Histogram window provides one option Scales gt 200 BE e 100 0 10000 20000 S0000 40000 532 W2 Mean Bkg GL Figure 2 33 Schematic Histogram Window Shortcut Menu The Scales option allows you to configure the graphical output After selecting the Scales option the Date Scaling dialog opens E Data Scaling E 1 X 532 W7 Mean Bkg aL Minimu
98. ox and Protocol Print Toolbar Using Microarray ol Print Options for Probe Table Clicking the Probe Table button switches to the layout setting controls for the Probe Table window To select the paper orientation Portrait or Landscape click the appropriate radio button in the Format section In the Print section the scope for the print output can be specified To enable printing of the table check the Table box and specify the appropriate output extent using the Full Table Rows above radio buttons Fratacnol Print Frint Preview 35 Probe Table Format Portrait C Landscape Print Table Full Table C Rows above 476 Figure 2 61 Array Analysis Table Print Options 32 AIDA Microarray User s Manual Print Options for Array Scatter Plot The Array Scatter Plot button on the Protocol Print toolbar allows you to configure the layout setting for the Array Scatter Plot window You can select the paper orientation Portrait or Landscape by clicking the appropriate radio button in the Format section The Graphic option in the Print section enables or suppresses the print output of the graphic displayed in the Array Scatter Plot window Protocal Print Print Preview Array Scatter Plot Format Portrait Landscape Print Graphic Figure 2 62 Array Scatter Plot Print Options Using Microarray 53 Print Options for Probe Scatter Plot The Probe Scatter Plot button
99. rrays can have up to three array levels In the example below the array is comprised of three levels a first level containing 3x2 fields a second level with 16x24 fields and a third spotting level with 4x4 dots Figure 3 21 Large Array Structure 90 AIDA Microarray User s Manual Defining a Field Matrix Array To define the first array level the surrounding frame overall mesh structure and repetition pattern select the New Evaluation item from the File menu All previous array overlays are cleared Array Definition Settings To define the geometry and labeling of the field matrix 1 Click the Edit Array Fields button on the Grid Definition panel of the Array Definition toolbox The appearance of the Grid Definition panel changes to reveal controls for specifying the number of columns Columns and rows Rows spacing between columns Y Spacing and rows X Spacing as well as their respective labeling In addition the Repetition Pattern Definition section of the pane allows you to specify repeated fields in the array 2 To divide the total array area into sub sections enter appropriate values in the Rows and Columns spin boxes Array Definition Grid Definition Repetition Pattern Definition Section Grid Geometry Definition Section Labeling Section OK Cancel Figure 3 22 Grid Definition Panel 3 To introduce spacing between sub sections enter appropriate length unit values in the X Spac
100. te are imported in activated and changeable mode You can adjust their position and resize or rotate these overlays before they are applied to the image see Step 3 Adjusting the Template to the Image for detail AIDA Microarray User s Manual Step 3 Adjusting the Template to the Image To adjust an imported template 1 In the Array Template panel of the Array Measurement toolbox which appears directly after loading a template enter the appropriate values in the spin boxes AND OR edit the template overlay directly by dragging the overlay to the desired position The Array Template panel contains spin boxes for fine tuning the adjustment of the array template in terms of array overlay x and y offset image and array resolution as well as spot diameter Hn Array Measurement Array Template x Offset ggsum y Offset 1 495um Image Res 5um Array Res 5um Spot Diameter 100um ok Figure 3 5 Array Template Adjustment Controls Setting the Image Res or Array Res parameters can be necessary if the scanning device used to acquire the image didn t embed the pixel resolution date for the image in the image file In this case the value in the template are interpreted as pixel values This can lead to misjudgments If you know the resolution of the image you can enter it in the Image Res spin box to circumvent this problem In addition you can adjust the spot diameter if necessary us
101. ter t a 5 193 1618 gt automatic full Xt XB eX tX Gamma ee XXX XX i Xt t ttt XN XXL m KI E 2930L 1375QL A3N4 38 502 8 OL Figure 2 30 Interlinked Windows Using Microarray 29 Schematic Histogram Window This data window shows a graphical representation of the distribution of values for a given data type selected via the controls of the Schematic Display panel on the Array Schematic toolbox as displayed in the Schematic Display window Schematic Histogram c Examples Examples Micr m 300 200 2 e 100 Uae 0 10000 20000 30000 40000 532 Mean Bkg GL Figure 2 31 Schematic Histogram Wind
102. the Array Tables AIDA s Automatic Export option allows you to export all array tables Array Result Table Analysis Table Probe Table automatically when you save an evaluation You can specify preferences for the range of the table columns to be automatically exported as well as export settings which are independent from the current work configurations of the table s The naming of the export files follows the scheme Evaluation Name Array Result Table txt Evaluation Name Analysis Table txt or Evaluation Name Probe Table txt that is if the name of the evaluation file is example adf the Array Result export file has the name example Array Result Table txt the Analysis Table export file is named example Array Result Table txtand the Probe Table export file example Probe Table txt Existing export files are overwritten without prompting the user However a backup file of the previous export file version is created Automatic Export always exports the entire table Restricting the export to selected rows is not possible The enabling of the automatic export option as well as its settings are evaluation specific attributes which are valid until they are explicitly changed using the Preferences or Automatic Export options of the Options menu or by importing an AIDA template If you create a new evaluation the current default configuration for Automatic Export is applied and will be saved with the evaluation data in the file
103. the Import by Global Label method is selected Specify the position of the label column in the source table using the Label Column spin box Import Name Table nco c adf Delimiter lt gt Li alr Label Column Figure 3 77 Specifying Label Column Position for the Global Label Method 130 AIDA Microarray User s Manual If the Import by Hierarchic Label method is selected e Specify the position of the label column in the source table using the 1 Level Label 2 Level Label Spot Label spin boxes Import Name Table nco c adf Delimiter lt gt 1 Level Label 1 2 Level Label EN BL Figure 3 78 Specifying Label Column Position for the Hierarchic Label Method Spot Label gt If the Import by Global Position method is selected e Specify the positions of the column and row indices in the source table using the Column Index and Row Index spin boxes Import Name Table nco c adf Delimiter lt gt Column Index Row Index Figure 3 79 Specifying Index Positions for the Global Position Method Performing Microarray Evaluations If the Import by Hierarchic Positions method is selected e Specify the positions of the column and row indices in the source table using the Column and Row spin boxes for 1 Level 2 Level Spot Level Import Name Table nco c adf Delimiter lt gt Li C Row T 1 Level 2 E
104. tion controls of the Array Definition toolbox The definition process consists of up to three steps according to the number of possible hierarchic array levels Array Levels Array overlays consist of up to three levels which can be selected for individual editing The simplest array structure inherits only the spotting matrix It consists of a first level with 1x1 fields and a third level comprised of 20x18 dots Ik PF OOO oOoooooscoomoes zDOOO0OMOoooOoOo9gsoOo wo sboo6oooooooeecoeooo aDOODOoOoooOoooosooooooh spoooooooooooooooos Korolo boooo amp ooooocOoOoOoOSoQg eooooOoooooOoOoOoooOoOoet sc oooooooooOoooQooD o oDOoooooooocOoOOOWc ovo uooooooesooooooooom eco coOoOoOoOoOoooo9s ooooooomweo slbooooooooocooooos doooooooooOo eooooootdunm slooooooooooooo0co0Q0dO tI 17 ogc eoo Nes CM He o oo OO Figure 3 19 Simple Array Structure Example Performing Microarray Evaluations 89 The most common structure consists out of two levels containing the surrounding frame with the blocks 16x24 fields and the spotting matrix 5x5 spots in each block respectively TUR 9 n rmm fem a teo re li ele ae TEM Sa C oi omar Rc ioo top l Figure 3 20 Most Common Array Structure Large a
105. to enable automatic segmentation of the spots If the restrict to selection option is enabled the automated alignment process 15 applied to selected part of the array only See the Standard Evaluation Procedure chapter for further information The Align button is used to start the alignment process The Reset button restores the selected part of the array to its initial state To undo the alignment choose the Undo command from the Edit menu Using Microarray 4 Rating Dot Values Section The quality of spot results can be influenced by a large number of factors for example spotting accuracy existence of artefacts overshining effects The Rating Dot Values section of the Array Measurement toolbox allows you to identify spots with problematic integration values manually Rating Dat values Channel Selection Menu Bad Spot Selection Tool Bad Spot Deselection Tool Unflag Bad Spots Button Flag as Bad Spot Button Error Code 1 Error Code Spin Box Unflag OK Spots Button Flag as OK Spot Button OK Spot Deselection Tool OK Spot Selection Tool Figure 2 46 Rating Dot Values Section The Rating Dot Values section contains the following controls The channel selection menu is used to select the channel for dot rating The Bad Spot Selection tool is used to flag the integration value of a spots as not reliable Just select the Bad Spot Selection tool and click the spot you want to mark
106. ue and if ratio value lt 1 the expression value is set to ratio value Note For values which are within the range of the expression thresholds represented by the non yellow colored lines and determined by the data range Min Expression spin box settings on the Ratio Comparison panel the real ratio values are calculated in any case This is due to the fact that small fluctuations in the measurement data causes large differences in interpretation especially at the intersection point of the Master Cut Off and the Client Cut Off For values which are within the range of the Max Ratio and Max Ratio thresholds the real ratio values are calculated and displayed in the Ratio column of the result table but the Expression value is set to On or Off Max Ratio Client Normalized Bkg 96 1 Max Ratio 0 0001 0 001 0 01 01 1 10 100 1000 10000 Master Normalized Bkg 2 Figure 3 72 Scatter Plot Partitioned Into Different Ratio Calculation Areas 126 AIDA Microarray User s Manual ay sy G y Client Normalized Bkg 1 Master Normalized Bkg Figure 3 73 Scatter Plot Partitioned Into Different Expression Value Calculation Areas Performing Microarray Evaluations 127 Importing Name Tables The Name Table Import option is intended for easy import of names and ID numbers of samples like clones sequences oligos and so on In Microarray you can import up to four columns from a source name table In ord
107. ue of the sum of the spots in each block Then each spot in the block is moved to a position with minimal circulare intensity fluctuations After the positioning process is completed outlier spots are determined and then included in the grid structure 80 AIDA Microarray User s Manual If Adaptive Diameter Segmentation is selected the diamater of each spot is determined by minimizing the distance between signal and spot center and the distance between background and the spot s peripherial region The automatic alignment and calculation process matches the position of the defined array spots with the position of the real dots in the array and performs all remaining measurement calculations background subtraction and so on The intensity values of all spots which have been aligned and integrated can be corrected for background As described in the respective section of this manual you can select the appropriate background subtraction method from the drop down menu of the Settings section of the Array Definition toolbox The subtraction the background values from each spot is performed automatically by the Alignment amp Calculation procedure The procedure tests guide dots to be in maximum paired dots to have almost equal intensities and empty dots to be in minimum If no spotting pattern is available or the use spotting pattern option is switched off the spotting matrices are moved to the nearest maximum In the second step of the a
108. ult Table Columns Columns Block Spot Row Index Column Index Field Row Field Column Block Row Block Column Spot Row Spot Column X pixel Y pixel Diameter pixel Area pixel Type Name ID unused 1 gt Short Description Block label potential reference column for name table import Spot label potential reference column for name table import Global grid position of a dot row component potential reference column for name table import Global grid position of a dot column component potential reference column for name table import Row position of the field within its array potential reference column for name table import Column position of the field within its array potential reference column for name table import Row position of the block within its field potential reference column for name table import Column position of the block within its field potential reference column for name table import Row position of the spot within its block potential reference column for name table import Column position of the spot within its block potential reference column for name table import X position of the spot measured of the left edge of the image Y position of the spot measured of the upper edge of the image Diameter of the spot Number of pixels of the spot Type of spot empty guide reference background Name of the probe to which the spot belongs column name can be
109. und Center In addition you can move the center of the array overlay using the mouse thereby moving its pivot point 12 0 15 dE dp EH iion mp Figure 3 14 00 TEE Center Performing Microarray Evaluations 85 Figure 3 15 Rotating Array Overlay Around Moved Center Since the array overlay center s pivot point function is limited to rotation task performed by dragging handles located at the edges of the overlay moving the center does not affect rotation tasks performed by dragging corner handles NS woe 025084109010 11712243 14 15 15 2 0 22 23 24 i 3 Sin c il 4 6779 siTe eru SW 1 x a WT NIS E i Ln x eis es a i e lt 7 if rd 3 F e LEGO F ehe ts 2525 Rigas 0 Figure 3 16 Rotating Array Overlay Using Corner Handle Center Moved 86 AIDA Microarray User s Manual pi ERES Confirm your modification by clicking the right mouse button or alternatively pressing the Return key 4 Right click to apply the changes Adjusting the position of particular dots In addition to editing the fields and blocks overlay geometry and position you can adjust the position of particular spots and or change their diameter To adjust the position of a spot 1 Double click the spot which you want to reposition to swit
110. und dots selected dots are interpolated for background subtraction Consequently the background subtraction is fitted to a locally varying background situation The influence of each background dot on any dot 15 weighted by the distance between those two dots As for the global background dots method if the use empty dots option is enabled the spots which are defined as empty in the spotting pattern are used for background subtraction Grid Background Dots The use of the selected dots for background subtraction is restricted to the sub grid where they are located The Mean or the Median values of the selected background dots flagged as Bkg or within a sub grid are averaged and then applied as background to all dots of the sub grid Lowest Grid Dots If this method is selected the dots with lowest Mean or Median values within a sub grid are taken for local background subtraction They are averaged and then applied to all dots of the sub grid Performing Microarray Evaluations 111 Specify the number of minimum intensity dots to be used for background subtraction in the Take Lowest spin box Take Lowest 1dats Bydot Mean Median Figure 3 55 Specifying Lowest Dots Local Dot Ring Background can also be subtracted directly around the overlay spot This results in an individual background determination of each subjected spot Inflate Dots O01 0 Ring width
111. xtent using the Full Table Rows above radio buttons Fratacnol Print ft tie Print Preview ER Analysis Table Format Portrait C Landscape Print Table Full Table C Rows above Figure 2 67 Analysis Table Print Options 58 AIDA Microarray User s Manual Print In AIDA there are two ways for printing your image and evaluation data 1 Select the Print option from the File menu or 2 use the Protocol Print section on the Publishing Layout toolbox The Print command of the File menu allows you to print your data on the fly without permanently storing the layout printing settings whereas the Protocol Print panel provides you with a complete set of printing features which includes saving the setting with the data file for reasons of reproducibility Selecting the Print option from the File menu opens the Print Microarray dialog allowing you to configure the printout to contain only the overlays results and graphs that are currently needed Print Microarray 2004 04 29 E X Printer Kyocera Mita FS 1010 KX at Megi Cancel Edges Left 2 0em E cm Header Printer Right 2 0cm Bottom 2 0cm Parameter Parameter and Image Options Image whole image overlays Probe Table portrait grayscale scales Array Scatter Plot automatic size v Probe Scatter Plot e 2 overexposed

AIDA Microarray for Windows User's Manual

Contents

Download Pdf Manuals

Related Search

Related Contents