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Lucidea Microarray ScoreCard

1. Layout Blanks Level 1 No Interval Display m Labels ma lal Rows 1244 275 pm E Measures y j Bakai ag E Columns z2 273 wm References E Alignment m Spot parameters Anchors MIETE raea oe Post analysis Oo aj OF 200 um Data channel File Refresh Cancel Help Figure 5 3 The Layout pane in the Protocol Editor window displaying the Level 1 array layout parameters In the Layout pane 1 Inthe Template organization area select Level 2 Spot groups N ote that Level 1 Spots should already be selected 2 IntheLevel 1 area make sure the number of rows is 12 or fewer and the number of columns is 32 3 Click the up arrow in the Level 1 area icon The Level 2 layout information appears figure 5 4 4 Inthe Level 2 area make sure the number of rows is 12 and the number of columns is 1 5 Select the remaining parameters that are best suited for your experiment E p5 4 Lucidea Microarray ScoreCard User s Guide 5 6 Setting the spot labeling method Protocol Editor xi Protocol Copy of md 1 Overview Analysis Images Layout Blanks Display E Labels Measures Background References E Alignment Anchors Post analysis T m r Template organization p C Level 3 Sub arrays aga na ee M Level 2 Spot groups F Level 1 Spots Level 2 No Interval _ ee Cl Rows 124 4452 um Columns 14 um i r Spot parameters Oo
2. even if the saturated pixels occur in one channel only B 2 Detecting the saturated pixels You can use ImageQ uant to detect saturated pixels When you view the image use the Grey Color Adjust feature to change the low end limit of the gray scale to 99998 and click OK The spots containing saturated pixels will appear black in the image Lucidea Microarray ScoreCard User s Guide pB 1 Appendix B Using the Amersham Pharmacia Biotech reflective slides with Lucidea Microarray ScoreCard S B 3 Eliminating the saturated pixels Amersham Pharmacia Biotech recommends that you choose one of the following methods to eliminate saturated pixels in your images Reduce the PM T setting Increased signals can saturate the scanner s PM T and produce saturated pixels Therefore reducing the PMT voltage setting can reduce or eliminate the pixel saturation problem Decrease the PM T voltage setting from the default value of 700 V to approximately 500 V Decreasing the PM T setting will not alter the detection limits the detection of low expressors because the background signals will also be reduced Reduce the amount of dye used per slide If pixel saturation occurs for both M icroarray ScoreC ard housekeeping genes and non ScoreC ard targets reduce the amount of the dye labeled probe used per slide Amersham Pharmacia Biotech recommends using 10 15 pmol of each dye per slide Reduce the amount of spike mix If pixel saturation occurs o
3. 2 Calculating the uncorrected log ratio 00005 A 3 A 2 3 Calculating the normalized log ratios 000 A 3 A 2 4 Adjusting the normalized log ratios 00000 A 3 A 2 5 Verifying the normalized results 0 0000 eee eee A 4 A 3 Estimating the confidence intervals for the gene expression data A 4 A 4 Calculating the default threshold for the mean NLR SD A 5 Appendix B Using the Amersham Pharmacia Biotech reflective slides with Lucidea Microarray ScoreCard B 1 Effects of the reflective slide on data normalization B 1 B 2 Detecting the saturated pixels 20 0 cece ee B 1 B 3 Eliminating the saturated pixels 00 00sec eee eee B 2 Glossary Lucidea Microarray ScoreCard User s Guide vii Preface About this guide The Lucidea M icroarray ScoreCard User s Guide explains how to use Lucidea M icroarray ScoreCard v1 1 so that accurate comparisons can be made within an experiment and across multiple experiments A PDF version of this guide is provided The PDF file resides in the M olecular D ynamics M icroarray M icroarray ScoreCard folder The software is provided with the control plate for processing the control plate data A Help file for the software is available To access the H elp file in the software choose H elp Topics from the H elp menu Related publications In addition to the Lucidea Microarray ScoreCard U ser s Guide the
4. 5 Actin Gene Performance table 0 0 00 eee eee eee 8 6 8 6 Normalized control plate data 2 eee eee 8 6 vi Lucidea Microarray ScoreCard User s Guide Table of contents 8 7 System validation information 0 0 0 a 8 9 8 7 1 Pen to pen variability 0 ee 8 9 8 7 2 Spot set to spot set variability 000005 8 10 8 8 Quality report file content 00 00 c eee ee 8 11 8 9 Normalized data file content 0 00 c cece eee 8 13 Chapter 9 Comparing data from multiple slides 9 1 Assessing data quality and statistical significance 00 9 1 9 1 1 Signal level and precision 00 0 cece eee 9 1 9 1 2 Sensitivity and Specificity 0 0 eee eee 9 2 9 1 3 Gene Expression data Accuracy and Precision 9 2 9 2 Interpreting the Gene Expression data from single and multiple hybridizations 20 2 eee 9 2 9 2 1 Calculating gene expression ratios 00 0000 9 3 9 2 2 Combining gene expression ratios from multiple spots Within a Slides Fed cep eiia Seance te Rac eaa Ried ac bi dia d 9 3 9 2 3 Combining data from replicate slides 0 9 3 9 2 4 Comparing data using the common reference approach 9 4 Appendix A Data normalization and statistical significance A 1 About the normalization method 00 0 0 e eee eee A 1 A 2 Normalizing the data 0 0 eee ee A 2 A 2 1 Working with the data in logarithmic scale A 2 A 2
5. For the single file output setting choose Total of channels 4 Total of views 1 or 4 e For the four file output setting G en3D B compatible Total of channels 1 Total of views 1 4 3 Retrieving the image files You should retrieve all four image files associated with the slide When you retrieve theimage files you must retrieve them in the order they were generated from the Generation III Array Scanner section 4 1 e L1 Spot set 1 left channel 1 e L2 Spot set 1 left channel 2 e R1 Spot set 2 right channel 1 e R2 Spot set 2 right channel 2 p4 2 Lucidea Microarray ScoreCard User s Guide 4 4 Selecting the spot labeling method 4 4 Selecting the spot labeling method The Lucidea M icroarray ScoreC ard software requires that the array elements be ordered in the horizontal sequence in the image analysis file figure 4 1 In ArrayVision when you create an array template in the Template Definition window be sure to choose the Sequential H orizontal labeling method O O O O O5 O O O O gt Figure 4 1 The sequential horizontal order 4 5 Selecting the background removal method When you set up the analysis for background removal you can select any location for background removal methods except the Selected Array Elements option You select the background removal method when you define the array template 4 6 Defining the references A reference is
6. Gls Data channel File v Refresh Cancel Help Figure 5 4 Setting Level 2 array layout parameters in the Layout pane 5 6 Setting the spot labeling method From the navigation pane choose Labels under Layout The Labels pane appears figure 5 5 Protocol Editor xi Protocol Copy of md 1 Overview E Analysis Images E Layout Blanks Display Display E Measures Background References E Alignment Anchors Post analysis p Label type Use custom labels Use structured labels E Level 3 Sub arrays O Level 2 Spot groups F Level 1 Spots Not verified an Gea Ga File ov Figure 5 5 Refresh Cancel Help Setting the spot labeling method in the Labels pane Lucidea Microarray ScoreCard User s Guide p5 5 Chapter 5 Preparing ArrayVision 5 1 analysis files for import SaaS In the Labels pane 1 In the Label type area select U se structured labels 2 Deselect Level 2 Spot groups N ote that Level 1 is preselected 3 Click Edit to define the labels The Label Editor window appears figure 5 6 4 In theLabel Editor window click the Auto number spots button point to Autosequence Row wise and choose N umeric labels 1 2 The Label Editor window displays the correct spot sequence for Lucidea M icroarray ScoreCard figure 5 6 Auto number spots button ial ajmh yaja as app af a
7. ScoreCard software To make sure you have set up the analysis correctly compare your exported file with the example input file in the M icroarray folder p5 8 Lucidea Microarray ScoreCard User s Guide Chapter 6 Preparing GenePix Pro analysis files for import This chapter describes the analysis settings that must be used in GenePix Pro 3 x to generate output that can be imported into the Lucidea M icroarray ScoreCard software The topics in this chapter are e Theimage and analysis data files section 6 1 Opening the image files in GenePix Pro section 6 2 e Selecting the feature layout section 6 3 e Saving the results file section 6 4 Depending on which 3 x version of GenePix Pro you are using the exact method for setting up the analysis can vary This chapter provides general guidelines required for the correct analysis output For detailed instructions on how to set up the analysis refer to the G enePix Pro 3 x Microarray Acquisition and Analysis Software U ser s G uide N ote An example analysis file generated by G enePix Pro 3 x isin the M icroarray folder 6 1 The image and analysis data files In two color microarray experiments you can use the GenePix 4000B M icroscanner to scan a slide and produce image files in a number of different ways As long as you scan the 12 pen areas and both spot sets on the slide you can produce the image files and the analysis results that are required by the Lucide
8. are added in the Cy3 or Cy5 labeling reactions Table 2 2 shows the concentrations and the relative abundance compared to MRNA in the labeling reactions of the spike mixes that correspond to the dynamic range control elements and the ratio control elements in the control plate These values can be used as quality measures that help you assess the output data in the software p2 4 Lucidea Microarray ScoreCard User s Guide Important Caution 2 3 Storing the control plate Table 2 2 Spike mix composition Conc in mix pg 5pl mix Relative Control sample Cy3 Cy5 Ratio Cy3 Cy5 abundance 1DR 1 1 33 000 33 000 3 3 2DR 1 1 10 000 10 000 1 3DR 1 1 1000 1000 0 1 4DR 1 1 330 330 0 033 5DR 1 1 100 100 0 01 6DR 1 1 33 33 0 0033 1RC 1 3 1000 3 000 NA 2RC 3 1 3 000 1000 NA 3RC 1 10 1000 10 000 NA 4RC 10 1 10 000 1000 NA For the labeling reactions add 5 ul of the appropriate spike mix per microgram of MRNA Use the spiked mRNA in the first strand cDNA synthesis reaction Thespiked mRNA can be labeled using oligo dT and or random primers If you plan to use the reflective slides from Amersham Pharmacia Biotech see appendix B forinstructions on howtoadjust the spike mix concentration to reduce pixel saturation and eliminate inaccurate data 2 3 Storing the control plate You can store the control plate for future experiments Before you storea used control plate cover the plate with a plastic
9. average signals 8 6 8 7 A thaliana 2 3 background removal method 4 3 5 7 bacterial genes 2 3 B subtilis 2 3 C comparative analysis 2 3 confidence intervals A 4 control plate 1 1 2 5 control samples 1 1 2 1 2 3 Cy3 Cy5 normalized log ratio 8 7 Cy3 Cy5 uncorrected log ratio 8 7 D defining references 4 3 5 8 detection limits 8 5 8 13 Detection Limits table 8 5 duplicate spotting pattern 2 6 dynamic range controls 2 4 evaluation 2 4 of experiment 8 1 values 8 5 Lucidea Microarray ScoreCard User s Guide Index 1 Index excluding references 4 3 exponential normalization A 1 exporting image analysis data 4 4 5 8 F feature layout 6 2 G GeneP ix 4000B image files 3 2 graph interpreting 8 1 H Help file 7 2 high density spotting mode 2 6 housekeeping gene function as control 2 3 performance 8 6 ratio 8 5 Housekeeping Gene Performance table 8 6 l image analysis exporting 4 4 5 8 output files 3 1 3 2 4 2 5 2 6 1 settings 4 3 5 3 image display format 4 2 image files Array Scanner 3 2 4 1 5 1 6 1 GenePix 4000B 3 2 retrieving 4 2 5 3 saving 6 3 input files 3 1 3 2 4 2 5 2 6 1 L loading image files in ArrayVision 5 3 logarithmic scale A 2 Index 2 Lucidea Microarray ScoreCard User s Guide M mean NLR SD 8 4 A 5 mean standard deviations comparing 8 7 Microarray ScoreCard describing 1 1 requirements 1 2 starting 7 1 workflow 1 3 microscanner settings 2 7 negative co
10. following publications are provided with the M icroarray System e Microarray System Applications G uide G ene E xpression Provides the latest information on protocols Contact Amersham Pharmacia Biotech for more information See Assistance for the contact information e Microarray System Training M anual Provides training material to be used during the training course Contact Amersham Pharmacia Biotech for more information See Assistance for the contact information e Microarray System Setup nstructions Provides instructions on how to set up the M icroarray System database and how to set up the computer monitor for the analysis workstation e Microarray System Generation III User s G uide Explains how to use Gen3DB the Array Spotter and the Array Scanner for microarray experiments e Microarray System ASC XT User s Guide for the Generation III Array Spotter Explains how to use the ASC XT software to produce higher density arrays with the Generation III Array Spotter instrument e Lucidea Automated Spotfinder Software H elp Explains how to use the Lucidea Automated Spotfinder to quantitate microarray images and produce analysis reports Lucidea Microarray ScoreCard User s Guide ix Preface Caution Important e ArrayVision O perations M anual Explains how to use the ArrayVision software to quantitate microarray images and produce analysis reports e GenePix 4000B Microarray Scanner U ser s
11. image analysis in ArrayVision you can produce one of the following e A single analysis file containing data from all four image files e A set of four individual analysis files each containing the data from one of the four image files compatible with Gen3DB The Lucidea M icroarray ScoreC ard software can analyze the data in either file type H owever because the software can process only files with specific field names and the fields must bein a particular order you must make sure that the analysis itself is set up properly for producing the correct output file 5 2 The Protocol Editor window In ArrayVision 5 1 you must set up the analysis protocol equivalent to the analysis template in version 4 x before you can begin the image analysis To set up an analysis protocol you must bein the Protocol Editor window figure 5 1 N ote that the Protocol Editor window is split into two panes e Navigation pane Displays an explorer tree e Display pane Displays the content of the item you choose from the explorer tree Navigation pane Display pane Figure 5 1 The Protocol Editor window p5 2 Lucidea Microarray ScoreCard User s Guide 5 3 Selecting the analysis parameters 5 3 Selecting the analysis parameters From the navigation pane figure 5 1 choose Analysis The Analysis pane appears In the Analysis pane In the Signal Type area select Fluorescent e In the Study Type area selec
12. in the Array Spotter e Normalized control plate data All the information from the data grid In addition for each control element the report shows the number of valid normalized log ratios used in the calculations Detection limits T he data from the D etection Limits table e Housekeeping gene performance T he data from the Actin Gene Performance table e Ratio analysis T he data from the Ratio Analysis table e System validation T he data from the System Validation window Normalization C onstants T he values used in the normalization calculations For information about the normalization calculation see appendix A e Mean 1 through 5DR NLS The mean normalized log ratios for the dynamic range control elements 1 through 5 This value is used to adjust the normalized log ratios for the dynamic range DR and ratio control RC elements Lucidea Microarray ScoreCard User s Guide p8 11 Chapter 8 Viewing the analysis results ca e a I Bicra Score ard Gusty Kapr I hipit File CUP rogpaen Fics Pb hee Lae Crp ec i Stray cb Tbe Dr pared BOL Gri Chanel Comn rii 10i 11 8 7 umber of Piai J Coertea Plate Pesii I Cotter Elereenbs Paca cv L ED 2a oE g SE 2 ie 35E BEDS Ersu a 187317 AE 1B SRE 1 53E0G Zi grz os TAS S ae IDET IIHI TWEE CET 20 ee INE iEn 125068 Gane ba SATIS AGO A610 AWE SHEDS mJ JihiEi u SI 05765 Oikia 7 10E00 gaias 6607 BEBU BEN 10 DRE
13. of quality N ote that the Lucidea M icroarray ScoreCard software does not evaluate the upper end of the dynamic range for deviations from linearity or the effects of saturated pixels The software highlights in red the dynamic range values that are less than the threshold you set The default threshold is 50 The example in figure 8 4 uses the default threshold and shows that all the dynamic range values are greater than the default threshold N ote If the dynamic range calculation results in the division by a 0 detection limit signal the software will display a value of 5 000 gt DL The percent of all signal values all samples including the control elements that are greater than the detection limits of the particular dye Low gt DL can indicate high background noise in the data or poor hybridizing sample quality A low gt DL can also indicate that you incorrectly specified the control plate position in the software Caution Besureto check the control plate position specification in the software before you evaluate the detection limit data Lucidea Microarray ScoreCard User s Guide p8 5 Chapter 8 Viewing the analysis results sl Detection Limits DL Channel isi ere DL Actin Ratio Dynamic Range gt DL cy3 16850 0 002 753 83 Cys 18749 0 014 1676 100 Figure 8 4 The Detection Limits table 8 5 Actin Gene Performance table Data in the Actin Gene Performance table figure 8 5 are calculate
14. or Windows 2000 and a minimum of 50 M B of free hard disk space e Internet Explorer 5 5 or higher e Two column arrays produced with the M olecular Dynamics M icroarray System Generation III Array Spotter running either the Generation II Array Spotter Control software or the ASC XT software e Lucidea Automated Spotfinder 1 0 or later analysis data in tab delimited text format e ArrayVision 4 0 or later analysis data in tab delimited text format e GenePix Pro 3 0 or later analysis data in the GenePix Pro results gpr format SSSA pl 2 Lucidea Microarray ScoreCard User s Guide Caution 1 3 Workflow overview 1 3 Workflow overview Figure 1 1 shows the overall workflow for the Lucidea M icroarray ScoreCard The image analysis files must be properly set up for input into the Lucidea Microarray ScoreCard software Be sure to follow the instructions in chapters 3 through 6 to correctly generate the image analysis data for the software For details on how to set up the analysis in Lucidea Automated Spotfinder ArrayVision or GenePix see the user documentation for the image analysis software Prepare the control plate for spotting section 2 4 y Set up the Microarray System Generation III Array Spotter and complete the spotting session section 2 5 Y Scan the slides with a Microarray System microscanner See the microscanner user documentation for detailed instructions Y Analyze the image files and export
15. relationship between the averaged C y3 signal values x axis and the averaged Cy5 signal values y axis for the following controls dynamic range ratio and housekeeping genes To see the signal values for a data point click the data point in the graph The signal values for that data point appear below the graph The graph provides an overall view of the dynamic range of the experiment and the performance of the ratio controls The plot of the dynamic range controls should appear approximately linear over three logs Under perfect experimental conditions the slope would be approximately 1 The example in figure 8 1 shows the relationship between the averaged Cy3 sDxA values x axis and the averaged Cy5 sDxA values y axis for the dynamic range ratio and housekeeping gene controls N otice that the dynamic range controls appear linear Lucidea Microarray ScoreCard User s Guide p8 1 Chapter 8 Viewing the analysis results Figure 8 1 The scatter plot of the control plate data in the Lucidea Microarray ScoreCard window p8 2 Lucidea Microarray ScoreCard User s Guide 8 2 Ratio Analysis table 8 2 Ratio Analysis table The Ratio Analysis table figure 8 2 allows you to compare the target ratios against the observed ratios for the control elements The table shows the following output data e Observed T he observed average ratio of the Cy3 signal to the Cy5 signal of the particular control element The value is obtain
16. the calculated value to which all array elements are normalized H owever do not define a reference using an array element because the software will exclude the array element from the exported data and misalign the remaining data 4 7 Defining the analysis settings When you define the analysis settings choose one of the following array types e For the single file output setting select M ultiple arrays and set the Array channels range from 1 to 4 e For the four file output setting Gen3DB compatible select Single array 4 8 Analyzing the image files After you finish setting up the parameters as described in this chapter analyze the image files using your usual process Lucidea Microarray ScoreCard User s Guide p4 3 Chapter 4 Preparing ArrayVision 4 x analysis files for import a 4 9 Exporting the analysis data After ArrayVision finishes the analysis export the analysis data to tab delimited text file s These files can be imported into the Lucidea M icroarray ScoreCard software To make sure you have set up the analysis correctly compare your exported file with the example input file in the M icroarray folder N ote that the example file was generated by ArrayVision 4 0 p4 4 Lucidea Microarray ScoreCard User s Guide Chapter 5 PreparingArrayVision5 1 analysis files for import This chapter describes the required analysis settings for ArrayVision 5 1 to generate output that can be imported into the Lucidea
17. 6 Normalized control plate data Cy3 CV The percent coefficient of variation for the Cy3 signal values Cy5 signal The average Cy5 signal values for replicates of the particular control element Cy5 CV The percent coefficient of variation for the Cy5 signal values Cy3 Cy5 Uncorrected Log Ratio T he average uncorrected log ratio of the Cy3 signal to the Cy5 signal of the particular contro element The log ratios for the dynamic range control elements and most housekeeping gene control elements should be close to 0 because no differential expression is expected for these controls H owever because of the differences in the Cy3 and Cy5 labeling reactions the uncorrected log ratios for these controls are frequently different than 0 In addition there is frequently a deviation from the constant log ratio at low signal values Cy3 Cy5 Normalized Log Ratio T he average normalized log ratio of the Cy3 signal to the Cy5 signal of the particular control element The normalized log ratios are calculated using a new proprietary method that considers signals from every array element not including the controls and compensates for differences in dye performance The normalized log ratio for the dynamic range control elements should be close to 0 You can use these values as references to compare data with high background signals artifacts systematic errors with probe labeling and other experiment errors Cy3 Cy5 SD for the Unc
18. 7 Using the Lucidea Microarray ScoreCard software R 7 2 Getting software help For instructions on how to use the Lucidea M icroarray ScoreCard software see the provided H elp To access the H elp choose H elp Topics from the H elp menu If you have questions about or require assistance with the software contact M icroarray System Technical Support See Assistance in the Preface for contact information p7 2 Lucidea Microarray ScoreCard User s Guide Caution Chapter 8 Viewing the analysis results This chapter explains how to view the analyzed data so that you can interpret the experiment results The topics in this chapter are e Control data scatter plot section 8 1 e Ratio Analysis table section 8 2 e Ratio Precision table section 8 3 Detection Limits table section 8 4 e Actin Gene Performance table section 8 5 Normalized control plate data section 8 6 e System validation information section 8 7 e Quality report file content section 8 8 e Normalized data file content section 8 9 For a detailed description of the calculations see appendix A Make sure you are using the correct settings in the Lucidea Microarray ScoreCard software before you evaluate the results N ote This chapter assumes that you are familiar with statistical and mathematical terms and concepts commonly used in scientific experiments 8 1 Control data scatter plot The control data scatter plot figure 8 1 shows the
19. Empty 2Reserved 20 D13 through D24 Reserved for future use Empty 3Reserved 21 E13 through E24 Reserved for future use Empty 4Reserved 22 F13 through F24 Housekeeping gene Est highly similar to NY REN 37 antigen 5HG Hs 173684 23 G13 through G24 Negative control Empty 3NC 24 H13 through H24 Housekeeping gene Human hydroxymethyl glutaryl CoA lyase 6HG Hs 831 EE p2 2 Lucidea Microarray ScoreCard User s Guide Table 2 1 Control samples 2 1 Control samples and functions and positions in a microplate continued Well set well positions ID in Control sample type Description ScoreCard 25 113 through 124 Housekeeping gene Neuroblastoma RAS viral oncogene homolog 7HG Hs 69855 26 J 13 through J24 Housekeeping gene Eukaryotic translation initiation factor 4A 8HG isoform 1 Hs 129673 27 K13 through K24 Housekeeping gene Ubiquinol cytochrome c reductase core 9HG protein II Hs 173554 28 L13 through L24 Housekeeping gene Enoyl Coenzyme A hydrolase short chain 1 10HG Hs 76394 29 M13 through M24 Housekeeping gene Actin gamma 1 Hs 14376 11HG 30 N13 through N24 Negative control Bacillus subtilis diaminopimelate 4NC decarboxylase lysA 31 013 through 024 Negative control Bacillus subtilis phenylalanine biosynthesis 5NC associated protein pheB 32 P13 through P24 Positive control Total human DNA complex target 2PC The total human DN A complex targets present in well sets 1 and 32 are go
20. G uide Explains how to use the GenePix 4000B microarray scanner to produce images of microarray slides e GenePix Pro 3 0 Microarray Acquisition and Analysis Software U ser s G uide Explains how to use the GenePix Pro 3 0 software to quantitate images of slides that are scanned on the GenePix 4000 microscanner e IQ Solutions Documentation Explains how to use the mageQ uant software to view and prepare the images for publication Special notices M ake sure you follow the precautionary statements presented in this guide Indicates that loss of data or invalid data could occur if the user fails to comply with the advice given Highlights information that is critical for optimal performance of the system N ote Identifies items of general interest Assumptions This guide assumes that you are familiar with the M olecular Dynamics Generation III M icroarray System the GenePix microscanner and one of the following image analysis software Lucidea Automated Spotfinder ArrayVision or GenePix Pro For information about the M icroarray System Lucidea Automated Spotfinder ArrayVision or the GenePix microscanner see the documents listed in the Related publications section This guide assumes that you have a basic understanding of the methods used in gene expression studies This guide also assumes that you have a basic understanding of mathematical terms and calculations commonly used in scientific experiments The
21. GB manual m 301 416 Lucidea Microarray ScoreCard User s Guide v1 1 part of Amersham Pharmacia Biotech Molecular Dynamics Cy ImageQuant Lucidea and ScoreCard are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia Corporation ArrayStat ArrayVision and Statistical Informatics are trademarks of Imaging Research Inc GeneP ix is a trademark of Axon Instruments Inc Microsoft Windows and Windows NT are trademarks of Microsoft Corporation Pentium is a trademark of Intel Corporation Spotfire Pro is a trademark of Spotfire Inc The Microarray System is for research purposes only It is not intended or approved for diagnosis of disease in humans or animals All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group that supplies them A copy of these terms and conditions is available on request Molecular Dynamics Inc 2001 All rights reserved April 2001 Limited license notice The Microarray ScoreCard and or its methods of use is covered by one or more pending US and foreign patent applications Nucleic acid arrays array makers systems instruments software and reagents are licensed for research use only No implied right to make use have made offer to sell lease distribute sell or import nuclei
22. M icroarray ScoreCard software The topics in this chapter are e Theimage and analysis data files section 5 1 e The Protocol Editor window section 5 2 e Selecting the analysis parameters section 5 3 e Loading the images section 5 4 Setting up the layout of the array section 5 5 Setting the spot labeling method section 5 6 e Selecting the analysis measures section 5 7 Selecting background subtraction methods section 5 8 e Defining the references optional section 5 9 e Analyzing the image files section 5 10 e Exporting the analysis data section 5 11 This chapter provides only the instructions required for the correct analysis output For detailed instructions on how to set up the analysis refer to the ArrayVision O perations M anual This chapter also provides analysis settings that are compatible with Gen3DB For more information about Gen3DB see the M icroarray System Generation III User s Guide N ote An example analysis file generated by ArrayVision 5 1 is in the M icroarray folder 5 1 The image and analysis data files In two color microarray experiments the Generation III Array Scanner produces four image files in the following order e Spot set 1 left dye 1 e Spot set 1 left dye 2 Spot set 2 right dye 1 Spot set 2 right dye 2 Lucidea Microarray ScoreCard User s Guide p5 1 Chapter 5 Preparing ArrayVision 5 1 analysis files for import SaaS When you perform
23. The Lucidea Microarray ScoreCard control reagents 2 1 Control samples and functions 1 00 0 0 cece eee 2 1 2 2 Spike mix composition and use 0 cece ee 2 4 2 3 Storing the control plate uaaa 2 5 2 4 Preparing the control plate for spotting 00 e eee 2 5 2 5 Setting up the Generation III Array Spotter 00 0 0 0 008 2 6 2 6 Setting up the microscanner 0 00 0 cc ee 2 7 2 7 Control element positions on aslide 1 2 eee 2 7 Chapter 3 Preparing Lucidea Automated Spotfinder analysis files for import 3 1 Image analysis requirements 0 ccc cee ee ees 3 1 3 2 Analyzing image files from the Array Scanner 0 000005 3 2 3 3 Analyzing image files from the GenePix 4000B Microscanner 3 2 3 4 Arranging the image analysis files 2 00 0 0 cc eee eee 3 2 Chapter 4 Preparing ArrayVision 4 x analysis files for import 4 1 The image and analysis data files 20 0 0 cece eee 4 1 4 2 Setting the display format a a aaa a 4 2 4 3 Retrieving the image files 2 0 0 cece eee 4 2 Lucidea Microarray ScoreCard User s Guide v Table of contents 4 4 Selecting the spot labeling method 00 00 cece 4 3 4 5 Selecting the background removal method 00005 4 3 4 6 Defining the references 0 0 c ec 4 3 4 7 Defining the analysis settings 0 0 0 0 cece eee ee 4 3 4 8 Analyzing the image files 00 0 0 cece eee 4 3 4 9 Exporting t
24. a M icroarray ScoreC ard software 6 2 Opening the image files in GenePix Pro To perform image analysis in GenePix Pro you must open all the image files associated with the slide The number of image files you open depends on how you scanned the slide and how many image files were produced As long as all of the images are opened when you perform analysis GenePix Pro will produce the required data for the Lucidea M icroarray ScoreCard software Lucidea Microarray ScoreCard User s Guide p6 1 Chapter 6 Preparing GenePix Pro analysis files for import E 6 3 Selecting the feature layout In GenePix Pro you define the array geometry before you begin the image analysis To define the array geometry open the N ew Blocks window figure 6 1 W hen you define the array geometry be sure to select R ectangular in the Feature Layout area xl Blocks Number of columns 2 Number of rows fi 2 Column spacing um fa500 4l Row spacing pm 4150 Column separation um 295 Row separation uml 545 Features TEA Number of columns 32 al ERDA t Rectangular soose Column spacing fum 280 4 vena aj C Glange packing Ht 33333 Number of rows fi 2 al t t C Orange packing 2 2323 Row specing ym 280 4 Feature diameter um ja D Rotation degrees fo 4 Cancel Help C Orange packing 3 weee C Drange packing 4 eese Figure 6 1 The New Blocks window p6 2 Lucide
25. a Microarray ScoreCard User s Guide 6 4 Saving the results file 6 4 Saving the results file After you finish the image analysis be sure to save the results in a single gpr format file figure 6 2 The Lucidea M icroarray ScoreCard software cannot open other formats from G enePix Pro Save in Sy overlay image files axon x t a EJ a 20um 32nm_635_nm gpr Filename 20um 532nm_635_nm Save as type GenePix Results Files gpr Cancel Comment Help Disk 3 628 GB free Naming I Date prefx I Numeric suffix I Barcode prefix IV Save a JPEG image containing all analyzed features Figure 6 2 The Save Results window Lucidea Microarray ScoreCard User s Guide p6 3 Chapter 7 Using the Lucidea Microarray ScoreCard software This chapter describes how to start the software and how to get instructions for the software The topics in this chapter are Starting the Lucidea M icroarray ScoreC ard software section 7 1 e Getting software help section 7 2 7 1 Starting the Lucidea Microarray ScoreCard software To start the software from the Windows desktop click Start point to Programs point to MD APPS point to Microarray and choose ScoreC ard The Lucidea M icroarray ScoreC ard window appears figure 7 1 Choose Help Topics to open the Help file Figure 7 1 The Lucidea Microarray ScoreCard window Lucidea Microarray ScoreCard User s Guide p7 1 Chapter
26. accurate data 2 7 Control element positions on a slide When you specify the normal spotting mode or the duplicate spotting pattern in the high density spotting mode each spot set includes the positive control elements negative control elements dynamic range control elements and ratio control elements Figure 2 1 shows an example of the placement of the control samples on a slide In this example e Thenormal spotting mode is specified in the Generation III Array Spotter Control software e Four plates are spotted four rows e Thecontrol samples are always in the first row of every pen area e The first and last control elements in the first row have the strong signals For detailed information about the N ormal spotting pattern see the Microarray System Generation 111 User s Guide Lucidea Microarray ScoreCard User s Guide p2 7 Chapter 2 The Lucidea Microarray ScoreCard control reagents Set1 Set 2 Duplicate 2 Al PoercsccccccccccccseseseeseeeeeS PeeccccccccccsccccesseseeseeeeeS 3 Al cccccccccccccccccccccccccccccccs coe ccccccccccccccccccccccccccece Plate 1 A2ie Laa a Control elements Bar code Figure 2 1 The control samples on a slide The Control_elements_imagetif file on the Lucidea M icroarray ScoreCard CD shows an array of control samples that are spotted repeatedly on the slide You can use this image to estimate the appearance signal strength co
27. an improve the confidence in the estimated standard deviation by using many replicate samples In addition you can pool variance estimates from many replicate groups assuming that the variance is the same among all the replicate groups With these principles you can calculate the confidence intervals based on the Z or normal distribution Z a x Mean NLR SD Cl Mean NLR Jn W here Cl is the confidence interval for the mean NLR for a given gene based on n replicates In the Generation III M icroarray System most genes are spotted in duplicate n 2 Cl specifies a range of values within which the mean NLR SD may lie You can calculate the intervals for different confidence levels For example a CI calculated at 99 5 is the range of values that will contain the true mean 99 5 of the time a is the probability of values falling outside the confidence interval because of random variation complements the confidence level for the mean NLR SD For example a 99 5 confidence interval has an value of 0 005 Z is a value that is a function of Based on a given value you can look up the corresponding Z value in a Z test table The Z value for an o of 0 005 is 2 807 N ote T he confidence interval equation assumes that the control samples are spotted in duplicate If one of the duplicates is not available drop the 2 from the equation A 4 Calculating the default threshold for the mean NLR SD A gene is significa
28. ation 1015117 10 1 7832674 24 4 1262281 20 6 lel Es 7786065 25 3 1307821 8262761 13 1 3 1380215 9567212 20 1 19 1574133 9186814 10 6 14 1461860 8490157 9 9 6 1376713 j 8052629 8 3 0 1461855 7781202 8 9 3 1396932 f 8039854 9 2 0 1477468 7961405 15 3 14 1404932 OPN B Bal spol an 7405791 47 6 8 1296313 Pai Set Spot Set ranon fetes Figure 8 7 The System Validation window 8 7 1 Pen to pen variability The first table in the System Validation window figure 8 7 shows pen to pen variability using the following data Pen Pen position in the pen assembly The pen assembly consists of 12 pens where pen 1 spots from well A 1 through P1 and A13 through P13 M ean For each pen the average of all Cy3 or Cy5 signal values from the actin gene in well sets 14 and 29 1HG and 11HG Lucidea Microarray ScoreCard User s Guide p8 9 Chapter 8 Viewing the analysis results Important e CV Percent coefficient of variation standard deviation mean of the average of all Cy3 or Cy5 signal values for the particular pen N ote that there are four replicates per pen per slide or a total of 48 elements per slide e Pen Variation Percent variation of a particular pen Mean Pen Meanactin Meanactin 100 M eanactin is the average actin gene signal as calculated in the Actin Gene Performance table section 8 5 The software highligh
29. base 10 of the N ormalized Cy3 signal to Cy5 signal ratio To calculate a gene expression ratio from a single spot take the antilog of Spot NLR by raising 10 to the power of Spot NLR Gene expression ratio from a single spot 10 SPotNLR This represents the ratio of the amount of the message in the Cy3 labeled mRNA to the amount of the message in the Cy5 labeled mRNA complementary to the individual spot For example if the Spot NLR value is 0 50 the gene expression ratio is 10 raised to the power 0 50 which is 3 16 T hus there is approximately a 3 16 fold more complementary message in the Cy3 labeled MRNA than in the Cy5 labeled MRNA If you want to report the ratio relative to the Cy3 labeled mRNA because the Cy3 labeled mRNA is your reference tissue reverse the sign of the NLR value The ratio becomes 10 to the power of negative Spot NLR Reverse ratio 10 SPotNLR 9 2 2 Combining gene expression ratios from multiple spots within a slide To combine data from multiple spots calculate the average normalized log ratio NLR values for each spot from a particular DN A sequence and raise 10 to the power of the average NLR Combined gene expression ratio 10 Average NLR for each spot Lucidea M icroarray ScoreC ard calculates and reports the average NLR for the duplicate spots in the normalized data file section 8 9 You should calculate the average value from the NLR before taking the antilog to transform to the combined g
30. c acid probe arrays or any other product in which Affymetrix or Molecular Dynamics has patent rights is conveyed by the sale of probe arrays instruments software or reagents hereunder This limited license permits only the use of the particular product s that the user has purchased from Affymetrix or licensed and sold by Molecular Dynamics or permitted licensees and may not be used in database generation for external license or sale or for service based pharmaceutical research Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech AB SE 751 84 Uppsala Sweden Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA Amersham Phamacia Biotech Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany Molecular Dynamics Inc 928 East Arques Avenue Sunnyvale CA 94085 4520 USA Table of contents Preface About this guide 2 0 2 eects ix Related publications 0 0 eee ix Special Mottes isis cashed pte eee ae ics Seas Se Gea E A xX AssUMPUONS s ii 53 244 Sas wa Saai Bend aa E eae a a aa oja xX ASS OE ote E RG title adel Sead xi Chapter 1 Introduction 1 1 About the Lucidea Microarray ScoreCard control plate 1 1 1 2 About the Lucidea Microarray ScoreCard software 1 1 1 3 Workflow overview 0 cece ete 1 3 1 4 Example files on the Lucidea Microarray ScoreCard CD 1 4 Chapter 2
31. d from the actin gene control element in well sets 14 and 29 This gene has the most replicates on the slide a total of 48 Therefore it can be a good indicator of system performance The Actin Gene Performance table shows the following output data e Signal T he average actin gene signal for the particular dye e SD The corresponding standard deviation value e CV The corresponding percent coefficient of variation which can indicate spot to spot variation and data reproducibility For example a high CV can indicate a damaged pen or high local background The software highlights in red the CV values that are greater than the threshold you set The default threshold value is 50 The example in figure 8 5 uses the default threshold and shows that all the CV values are less than the default threshold Actin Gene Performance Channel Signal SD CV I a 20 3 Cys 1367970 275378 20 1 Figure 8 5 The Actin Gene Performance table 8 6 Normalized control plate data The normalized control plate data grid figure 8 6 shows the following columns e Control The abbreviated name for the type of control used table 2 1 To see the associated clone used as the control rest the pointer on the control abbreviation and the clone information appears below the pointer e Cy3 signal The average C y3 signal values for replicates of the particular control element SaaS FT p8 6 Lucidea Microarray ScoreCard User s Guide 8
32. de DEES E 41379953 S437 D1 2B Ddi 400157 S505 IESE DSS IGI TRE OST Gee 3 wr HDA DS OE EA PA ete Me E e OTE E A Bidia M E ae em PE de Tc iarsi ES i SEQ A STH BME 317E 0EAN Sram 26062 WIE Sed ESS OSS OS 1223 200 ES 19 3655 D TST SME 013137 20065 51155 PAN ESA ALTE Od Oe a BiS MWO J Oe ee anz Sec I Aa VEE GEA COR GIAN 1a digi T GSH MIS S560 ARE Dede S755 S168 MMI IT OS Oe OAs 730837 066 ID 1356S Dae COS l Me SSSR E15 Bos Ier 080 JTBE 346E0e AFTI S 2 MUH HST JESN HET ISEE Hea oid riers AE Oa E 4S i e Aw Je OSS 016 iin 71514 2 p SES D5 07E EO a 7S AHi FT SPS OEB A PES 6366 46655 aF I 1H LISS OST BE 1 a uE ESE OSS NA TST EES ent RA WE aa TO OF DEING OR 160 EN Pe a CoE re 1550 77 ET 312300 Diaa OSes aT THENE EHE 0276155004 BET newn eho na Be Pe PES PE Ee PEPE Pe ee Re Figure 8 8 The quality report file in Excel Because of its size only the top half of the report is shown p8 12 Lucidea Microarray ScoreCard User s Guide 8 9 Normalized data file content Zo 8 9 Normalized data file content The normalized data file that you export contains the original image analysis data the uncorrected and normalized log ratio for every sample the normalized and mean values that were calculated for every sample and the clone information Figure 8 9 shows an example of a normalized file In this example the tab delimited text file is viewed in Excel for clarity DE me p yes HE h aro alld Baa ped r Sten Bae ra le
33. demonstrates this point the target 1RC value is 0 33 and the normalized value is 0 364 8 3 Ratio Precision table The Ratio Precision table figure 8 3 allows you to estimate the precision of the gene expression ratios in your experiment The table shows the following output data e Actin NLR SD Thestandard deviation of the normalized log ratio for the 48 replicate actin spots e Mean NLR SD The average standard deviation of the normalized log ratio for the following control types dynamic range DR ratio control RC housekeeping genes HG and positive control PC The standard deviation for each control is calculated from 24 replicates Ratio Precision Actin NLR SD Mean NLR SD 0 036 0 081 Figure 8 3 The Ratio Precision table Thetwo NLR SD values can indicate the reproducibility of the gene expression ratios for the hybridization experiment Smaller values for the NLR SD represent more precise gene expression ratios TheNLR SD values can also be used to estimate confidence intervals for the gene expression data For information about the calculation see appendix A The software highlights in red the M ean NLR SD value if it is greater than the threshold you set The default threshold value is 0 150 T he example in figure 8 3 uses the default threshold and shows that the M ean NLR SD is less than the default threshold To determine the threshold value to use and to understand how this value relates to the pr
34. e the software calculates the uncorrected log ratio of Cy3 signal to Cy5 signal as follows 1 For each spot the software calculates the log of the ratio of the Cy3 signal to the Cy5 signal The software does not calculate the log ratio if the Cy3 or the Cy5 signal is less than or equal to zero Log Cy3 signal Cy5 signal 2 The software calculates the log of the Cy5 signal Log Cy5 signal 3 After excluding all the spots from control plate elements the software finds the best fit constants a b and c for the following exponential normalization equation which is defined as the uncorrected log ratio ULR ULR a b exp log Cy5 signal c The software reports the best fit constants a b and c and the correlation coefficient R squared in the quality report For more information about the quality report see section 8 8 A 2 3 Calculating the normalized log ratios For each spot the software calculates the normalized log ratio NLR as follows NLR log Cy3 signal Cy5 signal a b exp log Cy5 signal c A 2 4 Adjusting the normalized log ratios Experiments have shown that when the ratio of the mRNA concentrations at the start of the labeling reaction is not 1 1 a constant offset results in the normalized log ratios for the dynamic range controls DR1 through DR6 and the ratio controls RC1 through RC4 To adjust for this offset the mean normalized log ratio for the dynamic range controls 1 throug
35. e 6DR relative abundance are not detectable 9 1 3 Gene Expression data Accuracy and Precision To determine the accuracy and precision of the ratios examine the Ratio Analysis table section 8 2 and the Ratio Precision table section 8 3 Consider the following when you inspect the information in these tables Inthe Ratio Precision table the average standard deviation of the normalized log ratio M ean NLR SD should be acceptable Typically the values should be less than the default threshold value of 0 150 e Inthe Ratio Analysis table the values of the normalized ratio and dynamic range controls should be acceptable based on your threshold difference from the target values e Thenormalized log ratios NLRs may be less accurate when one or more signals fall below the detection limit To assess the performance of the normalization process see appendix A 9 2 Interpreting the Gene Expression data from single and multiple hybridizations This section discusses how to use the output from Lucidea M icroarray ScoreC ard to obtain gene expression ratios n fold up regulation or down regulation and how to combine data across multiple hybridizations p9 2 Lucidea Microarray ScoreCard User s Guide Important 9 2 Interpreting the Gene Expression data from single and multiple hybridizations 9 2 1 Calculating gene expression ratios For each spot the Spot NLR valuein the normalized data file section 8 9 represents the log
36. ecision of the differentially expressed ratios see appendix A p8 4 Lucidea Microarray ScoreCard User s Guide 8 4 Detection Limits table ot 8 4 Detection Limits table The Detection Limits table figure 8 4 shows the following output data Detection Limit Signal T he average signal value of the plant derived negative control for Cy3 and for Cy5 plus 3 times standard deviation mean 3SD The detection limit data allows you to compare the detection limits across multiple slides within an experiment or across multiple experiments DL Actin Ratio The ratio of the detection limit to the average actin gene signal well sets 14 and 29 for the particular dye The detection limit to actin gene ratio measures sensitivity relative to the actin gene It can also be used to check detection limits from one or more experiments N ote that the smaller the ratio the greater the sensitivity The software highlights in red the values that are greater than the threshold you set The default threshold value is 0 025 The example in figure 8 4 uses the default threshold and shows that all the ratios are less than the default threshold Dynamic Range T he ratio of the maximum sDxA value to the detection limit signal for the particular dye The dynamic range data is an indicator of the quality of the data because higher dynamic range values mean lower background noise in the data H owever do not use the dynamic range data as an absolute measure
37. ed by taking the antilog of the average uncorrected log ratio section 8 6 e Normalized The observed average normalized ratio of the Cy3 signal to the Cy5 signal of the particular control element The value is obtained by taking the antilog of the average normalized log ratio section 8 6 Ratio Analysis Cy3 Cy5 Control Cy3iCys ARC 1 3 2RC 3 1 SRC 1 10 ARC 10 1 ADR 1 1 2DR 1 1 3DR 1 1 ADR 1 1 5DR 1 1 6DR 1 1 Figure 8 2 The Ratio Analysis table The software highlights in red the observed or normalized values if the fold difference between the observed or normalized value and the target value is greater than the threshold you set The default threshold setting is 1 5 fold difference The example in figure 8 2 uses the default threshold and shows that the fold difference between every observed value and its corresponding target value is greater than 1 5 Lucidea Microarray ScoreCard User s Guide p8 3 Chapter 8 Viewing the analysis results SSS Because of the differences in Cy3 and Cy5 the rate of incorporation stability and fluorescence the type of slide used and experimental errors the observed values do not necessarily match the corresponding target values On the other hand the normalization calculations should correct for these differences in the observed values Therefore the normalized values should closely match the corresponding target values The example in figure 8 2
38. eled skeletal muscle MRNA You can estimate the relative gene expression between brain and kidney from these data as follows Suppose the ratio of the average NLR for a given DNA sequence from the brain to the skeletal muscle hybridization is 1 23 Suppose the ratio of the average NLR for a given DNA sequence from the kidney to the skeletal muscle hybridization is 0 27 e TheNLR for brain over kidney can be calculated as the NLR for brain minus the NLR for kidney or 1 23 0 27 1 50 Raising 10 to the power 1 50 gives a brain to kidney gene expression ratio of 31 6 e In general the gene expression ratio of any condition to another when a common reference is used can be calculated from the following equation The normalized log ratio between two conditions NLR congitiona N LR condition2 The gene expression ratio 10 Rconditiont NLR conaition2 You should always interpret the gene expression ratios with caution if one or more of the signals used to calculate them fall below the detection limit p9 4 Lucidea Microarray ScoreCard User s Guide Appendix AData normalization and statistical significance This appendix describes the Lucidea M icroarray ScoreCard normalization method The topics in this appendix are About the normalization method section A 1 e Normalizing the data section A 2 e Estimating the confidence intervals for the gene expression data section A 3 e Calculating the default thre
39. ene expression ratio This also applies to calculating confidence intervals see section A 3 9 2 3 Combining data from replicate slides To combine data from multiple replicate slides average the NLR values for all the replicate spots of each DNA sequence before transforming the values from NLRs to gene expression ratios If you performed replicate hybridizations using the dye reversal method you have to correct for the dye reversal in your calculations In dye reversal experiments on the first slide the experimental RNA is labeled with Cy3 and the reference RNA is labeled with Cy5 and on the second slide the experimental RNA is labeled with Cy5 and the reference Lucidea Microarray ScoreCard User s Guide p9 3 Chapter 9 Comparing data from multiple slides Important RNA is labeled with Cy3 When you perform the calculations you have to correct for the dye reversal by reversing the sign of the NLR values for the second slide before calculating the average with the NLR values from the first slide Then you can transform the NLR values to the gene expression ratios as described in section 9 2 2 9 2 4 Comparingdata usingthe common reference approach You can compare data from the common reference RNA preparation for several hybridizations For example you can perform one experiment using Cy3 labeled brain mRNA and Cy5 labeled skeletal muscle MRNA In a second experiment the hybridization involves Cy3 labeled kidney mRNA and Cy5 lab
40. expressed in terms of percent CV Standard Deviation Mean 100 control elements the contro samples that are spotted on the specially coated microscope slides fluorescence the emission of light by a dye molecule as a result of the absorption of laser light gene expression the manifestation of a characteristic that is specified by a gene such as the production of a protein by a gene housekeeping genes ubiquitously expressed genes found in all cell types In a microarray experiment the housekeeping genes are those with minimal expression variation and are used for normalization of the samples hybridizing samples the single stranded DNA or RNA fragments that bind to the array elements in the hybridization experiments microarray an orderly arrangement of DNA or RNA fragments with a spot size approximately 100 400 um in diameter M icroarrays usually containing thousands of spots are used in high throughput gene mapping mutation detection studies and gene expression analysis Lucidea Microarray ScoreCard User s Guide Glossary 1 Glossary negative control the array element used for evaluating the degree of background signal levels of nonspecific hybridization normalization the process of calculating the ratio or the difference between a set of standard array element signal values and the sample signal values to bring the data to the same scale for comparative expression studies positive contr
41. gh A12 Positive control Total human DNA complex target 1PC 2 B1 through B12 Negative control Arabidopsis thaliana protein G1p INC 3 C1 through C12 Dynamic range control Yeast Intergenic Region from Chrom XI 1DR 4 D1 through D12 Dynamic range control Yeast Intergenic Region from Chrom XIV 2DR 5 E1 through E12 Dynamic range control Yeast Intergenic Region from Chrom VII 3DR 6 F1 through F12 Dynamic range control Yeast Intergenic Region from Chrom VII 4DR 7 G1 through G12 Dynamic range control Yeast Intergenic Region from Chrom XII 5DR 8 H1 through H12 Dynamic range control Yeast Intergenic Region from Chrom XII 6DR 9 I1 through 112 Ratio control Yeast Intergenic Region from Chrom XII IRC 10 J 1 through J 12 Ratio control Yeast Intergenic Region from Chrom XII 2RC 11 K1 through K12 Ratio control Yeast Intergenic Region from Chrom XII 3RC 12 L1 through L12 Ratio control Yeast Intergenic Region from Chrom XIII 4RC 13 M1 through M12 Negative control PolydA oligonucleotide 2NC 14 N1 through N12 Housekeeping gene Actin gamma 1 Hs 14376 1HG 15 O1 through 012 Housekeeping gene Glyceraldehyde 3 phosphate dehydrogenase 2HG Hs 169476 16 P1 through P12 Housekeeping gene Ubiquinol cytochrome c reductase core 3HG protein II Hs 173554 17 A13 through A24 Housekeeping gene Casein kinase II beta polypeptide Hs 165843 4HG 18 B13 through B24 Reserved for future use Empty 1Reserved 19 C13 through C24 Reserved for future use
42. h 5 DR1 through DR5 is subtracted from each dynamic range and ratio control normalized log ratio Adjusted NLR for a DR or RC NLR Mean NLR for DR1 to DR5 N ote T he normalized log ratio of dynamic range control 6 DR6 is not used in this calculation because it is frequently below the detection limit The software reports the average unadjusted NLR for DR1 5 in the quality report For more information about the quality report see section 8 8 Lucidea Microarray ScoreCard User s Guide pA 3 Appendix A Data normalization and statistical significance Lz A 2 5 Verifying the normalized results You can verify the normalized results as follows e After you specify the dye channel assignment in the software display the normalized control plate data in the data grid section 8 6 Compare the standard deviations of the average normalized log ratio NLR SD to the standard deviations of the average uncorrected log ratio ULR SD The exponential normalization method improves the precision performance of the replicate groups if the average NLR SD is less than the average ULR SD e After you specify the dyechannel assignment in the software verify that in the Ratio Analysis table the normalized values closely match the corresponding target values e After you specify the dyechannel assignment in the software examine the standard deviations of the average normalized log ratio NLR SD Reverse the dye channel assignment and observe
43. he analysis data 1 ee 4 4 Chapter 5 Preparing ArrayVision 5 1 analysis files for import 5 1 The image and analysis data files 00 cece eee eae 5 1 5 2 The Protocol Editor window 0 0 c cece eee 5 2 5 3 Selecting the analysis parameters 0 2 0 0c cece eee eee 5 3 5 4 Loading the images 0 cece ett 5 3 5 5 Setting up the layout of the array 6 eee 5 4 5 6 Setting the spot labeling method 0 0 000 cece eee 5 5 5 7 Selecting the analysis measures 0 0 cece eee 5 7 5 8 Selecting background subtraction methods 0e0eeeae 5 7 5 9 Defining the references optional 0 cee eee eee 5 8 5 10 Analyzing the image files 0 0 eee ee 5 8 5 11 Exporting the analysis data 0 2 eee eee 5 8 Chapter 6 Preparing GenePix Pro analysis files for import 6 1 The image and analysis data files 00 cee eee eae 6 1 6 2 Opening the image files in GenePix Pro s eee 6 1 6 3 Selecting the feature layout 0 2 eee 6 2 6 4 Saving the results file 2 ete 6 3 Chapter 7 Using the Lucidea Microarray ScoreCard software 7 1 Starting the Lucidea Microarray ScoreCard software 7 1 7 2 Getting software help 2 eae 7 2 Chapter 8 Viewing the analysis results 8 1 Control data scatter plot onua 8 1 8 2 Ratio Analysis table 0 0 eee 8 3 8 3 Ratio Precision table 2 ke eee 8 4 8 4 Detection Limits table 0 0 eee 8 5 8
44. i H Ei 1 w TH pr 2 f EH 25 HERS IIE ar kt amp i 7 ii 1 i Figure 5 6 The Label Editor window displaying the correct spot sequence for import into the Lucidea Microarray ScoreCard software p5 6 Lucidea Microarray ScoreCard User s Guide 5 7 Selecting the analysis measures 5 7 Selecting the analysis measures From the navigation pane choose M easures T he M easures pane appears figure 5 7 In the M easures pane 1 Choose Volume or Density from the Principle M easure list 2 Choose M ean or M edian from the Background list 3 Select any measure from the Name list Protocol Editor x Protocol Copy of md 1 Principal Tverview measure E Analysis Background Mean x Images E Layout Blanks Density Display J SD Standard Deviation E Labels C Area Measures L Position XY Background M Bkgd Background References Cl Ref or sRef Reference or Corrected Refere El Alignment C S N Signal to Noise Ratio Anchors sDens or nDens Corrected Density Post analysis O Flag M Diff C D C Diff D C x Note only available if defined File Refresh Cancel Help Figure 5 7 The Measures pane 5 8 Selecting background subtraction methods From the navigation pane choose Background under M easures The Background pane appears figure 5 8 In the Background pane you can select any of the available background subtraction me
45. lor and sequence of the contro elements on your slides p2 8 Lucidea Microarray ScoreCard User s Guide Chapter 3 Preparing Lucidea Automated Spotfinder analysis files for import This chapter describes the image analysis requirements for the Lucidea Automated Spotfinder to generate output that can be imported into the Lucidea M icroarray ScoreCard software The topics in this chapter are e Image analysis requirements section 3 1 e Analyzing image files from the Array Scanner section 3 2 e Analyzing image files from the GenePix 4000B M icroscanner section 3 3 e Arranging the image analysis files section 3 4 This chapter provides general guidelines required for the correct analysis output For detailed instructions on how to set up the analysis refer to the Lucidea Automated Spotfinder H elp 3 1 Image analysis requirements For two color microarray experiments Lucidea Automated Spotfinder requires four individual image files e Spot set 1 left dye 1 e Spot set 1 left dye 2 e Spot set 2 right dye 1 Spot set 2 right dye 2 Lucidea Automated Spotfinder produces a set of four individual analysis files each containing the data from one of the four image files The Lucidea M icroarray ScoreCard software can open these files directly and perform the calculations Lucidea Microarray ScoreCard User s Guide p3 1 Chapter 3 Preparing Lucidea Automated Spotfinder analysis files for import E 3 2 Ana
46. lyzing image files from the Array Scanner In two color microarray experiments the Generation III Array Scanner produces four image files in the following order e Spot set 1 left dye 1 e Spot set 1 left dye 2 Spot set 2 right dye 1 Spot set 2 right dye 2 These files match the requirements for Lucidea Automated Spotfinder Therefore you can scan the slides without using any special settings 3 3 Analyzing image files from the GenePix4000B Microscanner If you want to use Lucidea Automated Spotfinder to analyze the image files from the GenePix 4000B M icroscanner you need to set up the scan as follows to produce four separate image files Scan spot set 1 and spot set 2 independently e Scan each of the two dyes independently For instructions on how to scan a slide using the GenePix 4000B M icroscanner see the G enePix 4000B Microarray Scanner User s Guide 3 4 Arranging the image analysis files Lucidea Automated Spotfinder creates four analysis files that arein the tab delimited text format You need to keep these files in the same folder to import them into the Lucidea M icroarray ScoreCard software To automatically store the analysis files in the same folder after analysis in the Preferences window figure 3 1 select Results stored in default output directly in the Results Location area Alternatively you can move the four files into a new folder after you finish the analysis For information on h
47. n The plant gene is also used to determine the detection limit The polydA oligonucleotide in well set 13 does not lead to high levels of hybridization Therefore it is also a good negative control N C element for verifying the degree of nonspecific hybridization in an experiment In well sets 3 through 8 six sequences derived from intergenic regions in yeast have been shown not to hybridize with mammalian cDNAs Using a spike mix of the corresponding mRNA in the labeling reactions and hybridizing the mix on yeast targets produces signals that serve as dynamic range DR controls in the experiment section 2 2 Dynamic range controls allow you to evaluate the dynamic range and sensitivity of the system In well sets 9 through 12 four other sequences derived from intergenic regions in yeast have also been shown not to hybridize with mammalian cDNAs Using a spike mix of the corresponding mRNA in the labeling reactions and hybridizing the mix on yeast targets produces signals that serve as ratio controls RC in the experiment section 2 2 Ratio controls are references for measuring differential expression 2 2 Spike mix composition and use Avoid repeated freezing and thawing of the spike mixes Provided with the control plate are 20 tubes of spike mixes e 10 tubes each containing 500 ul of the Cy 3 spike mix e 10 tubes each containing 500 ul of the Cy5 spike mix For the dynamic range control and ratio control samples MRNA spikes
48. nactin is the average actin gene signal as calculated in the Actin Gene Performance table section 8 5 p8 10 Lucidea Microarray ScoreCard User s Guide 8 8 Quality report file content The software highlights in red all Spot Set Variability results with absolute values exceeding the threshold you set The default threshold setting is 10 For example if you use the default threshold for spot set variability all the results with absolute values exceeding the 10 threshold are highlighted in red Figure 8 7 does not show any value exceeding the default threshold for spot set variability If you see highlighted values in the table the highlights can indicate possible problems with slide surface chemistry and should be considered when you evaluate the data 8 8 Quality report file content The quality report that you export contains all the data you see in the Lucidea M icroarray ScoreCard window including the system validation data Figure 8 8 shows an example of a quality report file In this example the tab delimited text file is viewed in Excel for clarity The quality report shows the following information e Header information T he type of report file the number of input files the input file name s and location s the signal data processed the channel assignment of the Cy3 data comments about the analysis the date of the analysis the total number of microplates in the experiment and the position of the control plate
49. nly with the M icroarray ScoreCard dynamic range or ratio control targets use 60 of the recommended amount of spike mix to add to the labeling reaction Reducing the spike mix will not alter the detection limit or perturb the ratios When performing microarray experiments you should always review the results of each experiment to eliminate inaccurate data When you use M icroarray ScoreCard make sure you understand the outcome of the ScoreCard analysis pB 2 Lucidea Microarray ScoreCard User s Guide Glossary array elements the cloned DNA fragments or PCR products that are spotted on the specially coated microscope slides DxA density value of an array element multiplied by its area Also referred to as volume this measurement is the total amount of signal in an array element For more information about D xA see the ArrayVision O perations M anual or H elp file Dens density value for each spot The value represents the average of all the pixels contained in the spot For more information about Dens see the ArrayVision O perations M anual or Help file dynamic range control control elements used to evaluate the dynamic range and sensitivity of the system channel in ArrayVision a storage area for a single image Each image is associated with a dye used in the microarray experiment clone library a set of cloned DNA fragments coefficient of variation standard deviation divided by the mean often
50. ntly differentially expressed if the confidence interval for the M ean NLR does not include 0 Using the a value of 0 005 section A 3 you can approximate the significance in the difference in NLR by calculating the half width for the confidence interval Clpa Z Average NLR SD 2 Raise the Cly ar result to the power of 10 to convert the result from logarithmic space to n fold space Table A 2 lists the results of these calculations for an a value of 0 005 The calculations assume the controls are spotted in duplicate on the slide Lucidea Microarray ScoreCard User s Guide pA 5 Appendix A Data normalization and statistical significance Table A 2 Mean NLR SD values and the corresponding detectable changes Mean NLR SD Detectable Change n fold 0 050 1 26 0 100 1 60 0 150 1 98 0 200 2 49 0 250 3 13 Table A 2 shows that a two fold or greater change is considered significant when a is 0 005 and if the mean NLR SD is less than or equal to 0 150 The software uses this value as the default mean NLR SD threshold By selecting a different value a different level of gene expression difference to detect and a different number of replicates you can calculate a different threshold to set in the software N ote T he confidence intervals calculated in this section are based on the Z distribution and are good approximations You can use the ArrayStat Statistical Informatics software for the exact statis
51. ntrols 2 3 2 4 nonspecific hybridization 2 4 normal distribution A 5 normal spotting mode 2 6 normalization calculation A 1 normalized data grid 8 6 normalized log ratio 8 13 A 3 normalized signal ratio 8 3 8 13 0 observed signal ratio 8 3 P pen variability 8 9 pixels saturated B 1 plant genes 2 3 poly A sequences 2 4 positive controls 2 3 Protocol Editor window 5 2 Q quality report 8 11 R Ratio Analysis table 8 3 ratio controls description 2 4 ratio controls performance 8 1 Ratio Precision table 8 4 reflective slides 2 5 2 7 B 1 retrieving image files in ArrayVision 4 2 5 3 S saturated pixels B 1 saving image files in GenePix Pro 6 3 scanner settings 2 7 scatter plot interpreting 8 1 sensitivity evaluation 2 4 sequence of array elements 4 3 5 4 5 5 signal data 8 6 8 7 single plate spotting mode 2 6 Slides reflective 2 5 2 7 B 1 spike mix 2 4 Spotfire Pro software 8 14 spot set variability 8 10 spotting control samples 2 7 spotting mode high density 2 6 normal 2 6 single plate 2 6 spotting pattern duplicate 2 6 Statistical Informatics software 8 14 system validation 8 9 system verification 2 6 U uncorrected log ratio 8 3 8 13 A 3 Y yeast sequences 2 4 Lucidea Microarray ScoreCard User s Guide Index Index 3
52. od positive control PC elements because they always produce measurable signals regardless of the tissue specific mRNA used for hybridizing sample synthesis These positive controls are useful in ArrayVision analysis because their positive signals serve as grid anchors for array template creation They are also useful indicators of the quality of the hybridizing mRNA samples The various housekeeping genes H G present in well sets 14 through 17 and well sets 24 through 29 serve as additional positive control elements The expression of housekeeping genes is unlikely to fluctuate significantly within a given tissue Therefore they are used for the comparative analysis of the slides within a single experiment or across multiple experiments N ote T he housekeeping gene Actin gamma 1 is used twice in the control plate in well set 14 1H G and well set 29 11HG It is used to calculate the detection limit to actin ratio and the percent coefficient of variation CV for each dye For details about these calculations see chapter 6 The A thaliana protein G1p in well set 2 and the bacterial genes from B subtilis in well sets 30 and 31 are not present in human derived cDNAs R Lucidea Microarray ScoreCard User s Guide p2 3 Chapter 2 The Lucidea Microarray ScoreCard control reagents Important Therefore they are useful as negative control N C elements for evaluating the degree of the background signal levels of nonspecific hybridizatio
53. ol the array element used for the normalization of the samples quantitation the process in which signal intensities of the array spots and other values are calculated ratio control control elements that are used as reference for measuring differential expressions relative fluorescence units rfu the arbitrary units in which fluorescence intensity is reported by the Array Scanner SD standard deviation of the pixel density values For more information about SD see the ArrayVision O perations M anual or H elp file sD ens subtracted density value Density value of the spot minus the background density value For more information about sD ens see the ArrayVision O perations M anual or H elp file sD xA background subtracted DxA or an array element s total signal volume minus the background volume sDxA Densitydata Densitybkgd Areadata For more information about sDxA see the ArrayVision O perations M anual or H elp file spot buffer the distance between the edges of consecutive spots on the slide spot diameter the length of the chord that passes through the center of a circular region within which a spot will be placed The spot diameter added to the spot buffer produces the center to center distance between consecutive spots spot set a group of spots on a slide occupying an area covered by a single scan pass Each slide can have up to two spot sets spot set 1 is on the left side sp
54. or foil adhesive film Store the control plate at 20 C 2 4 Preparing the control plate for spotting Make sure you follow the instructions shipped with the Microarray ScoreCard control reagents for diluting and preparing the control samples for spotting To use the control plate m Remove the control plate from storage Leave the adhesive cover on the plate N Warm the plate to room temperature has Spin the plate at 3000 x g for 3 5 minutes Lucidea Microarray ScoreCard User s Guide p2 5 Chapter 2 The Lucidea Microarray ScoreCard control reagents Caution Important Important Amersham PharmaciaBiotech recommends alongspintocollectcondensatefrom the adhesive film and to prevent cross sample contamination 4 Peel off the adhesive cover 5 Prepare the control plate exactly as described in the instructions shipped with the M icroarray ScoreCard control reagents 6 Load the control plate into the Generation III Array Spotter instrument For instructions on how to properly load the microplate into the Array Spotter see the M icroarray System G eneration III User s Guide Tofacilitateimageanalysis Amersham PharmaciaBiotech recommends you place the control plate in slot 1 in the microplate elevator so that the control samples are placed in the first row of each pen area on the slides 7 Close the Array Spotter door s 2 5 Setting up the Generation III Array Spotter When you set up the spotting ses
55. orrected Log Ratio and N ormalized Log Ratio The standard deviations of the uncorrected log ratio and normalized log ratio values M ean The overall mean and the mean standard deviations of the uncorrected and normalized log ratios for the dynamic range ratio and housekeeping gene control elements These values appear at the bottom of the grid Comparing the mean standard deviations of the uncorrected and normalized signal ratios allows you to evaluate the normalization process The mean standard deviations provide an estimate of the precision of the log ratios and can be used to estimate confidence intervals for the gene expression ratios The mean standard deviations of the normalized log ratio should be less than that of the uncorrected log ratio If this is not the case check and make sure that you have the correct dye channel assignment in the software Lucidea Microarray ScoreCard User s Guide p8 7 Chapter 8 Viewing the analysis results Figure 8 6 The data grid in the Lucidea Microarray ScoreCard window Note that the grid in this example is color coded by the type of control element p8 8 Lucidea Microarray ScoreCard User s Guide 8 7 System validation information 8 7 System validation information You can use the information in the System Validation window figure 8 7 to check the following Pen to pen variability section 8 7 1 Spot set to spot set variability section 8 7 2 System alid
56. ot set 2 ison the right side In a normal mode spotting pattern the array elements in spot set 2 are the duplicates of spot set 1 spotting area the area of the slide within which the Array Spotter spots the DNA or RNA material The spotting area on the slide is approximately 18 36 mm by 54 25 mm Glossary 2 Lucidea Microarray ScoreCard User s Guide Glossary spotting mode the logical flow in which the Array Spotter spots the DNA or RNA material on the slides spotting pattern the pattern on the slide that results from a particular spotting mode sVOL subtracted volume value Volume value of the spot minus the background volume value For more information about sVOL see the ArrayVision O perations M anual or Help file VOL density value of each spot multiplied by its area For more information about VOL see the ArrayVision O perations M anual or Help file well set a group of 12 consecutive wells in a microplate Each well set corresponds to a set of wells spotted together by 12 spotting pens Each 384 well microplate contains 32 well sets Lucidea Microarray ScoreCard User s Guide Glossary 3 Index A actin NLR SD 8 4 analysis measures 5 7 analyzing image files 4 3 5 8 array element sequence for analysis 4 3 Array Scanner image files 3 2 4 1 5 1 6 1 ArrayStat Statistical Informatics software 8 14 ArrayVision analysis 2 3 4 1 5 1 6 1 output 1 2 Automated Spotfinding Application analysis 3 1
57. ow to use Lucidea Automated Spotfinder see the Lucidea Automated Spotfinder H elp p3 2 Lucidea Microarray ScoreCard User s Guide 3 4 Arranging the image analysis files Preferences Figure 3 1 The Preferences window Lucidea Microarray ScoreCard User s Guide p3 3 Chapter 4 Preparing ArrayVision 4 x analysis files for import This chapter describes the analysis settings that must be used in ArrayVision 4 x to generate output that can be imported into the Lucidea M icroarray ScoreCard software The topics in this chapter are e Theimage and analysis data files section 4 1 e Setting the display format section 4 2 e Retrieving the image files section 4 3 Selecting the spot labeling method section 4 4 Selecting the background removal method section 4 5 e Defining the references section 4 6 e Defining the analysis settings section 4 7 e Analyzing the image files section 4 8 e Exporting the analysis data section 4 9 Depending on which 4 x version of ArrayVision you are using the exact method for setting up the analysis can vary This chapter provides general guidelines required for the correct analysis output For detailed instructions on how to set up the analysis refer to the ArrayVision O perations M anual This chapter also provides analysis settings that are compatible with Gen3DB For more information about Gen3DB see the M icroarray System Generation III User s Guide N ote An e
58. ption of the data that the software generates see chapter 8 9 1 1 Signal level and precision To determine the reproducibility of signals within one channel you can use the signal level and precision information in the Actin Gene Performance table section 8 5 Smaller CV values for actin indicate more reproducible actin signals across the slide and more uniform background and hybridization If actin appears to be differentially expressed use another housekeeping gene such as enoyl coenzyme A hydrolase 10H G to determine the reproducibility of the signal levels across slides Lucidea Microarray ScoreCard User s Guide p9 1 Chapter 9 Comparing data from multiple slides 9 1 2 Sensitivity and Specificity To assess the system sensitivity and to check for non specific binding use the information in the Detection Limits table section 8 4 The detection limit signal information can indicate the sensitivity of the Microarray system and the overall quality of each hybridization e Thelower the signal detected the greater the sensitivity Thehigher the signal detected the lower the specificity of hybridization The dynamic range controls DRs correlate with the relative abundance of the mRNA spike and can indicate the expression level that can be detected For example if dynamic range 6 6DR is not above the threshold signal level for the detection limit you can state with confidence that genes with expression levels below th
59. rd User s Guide xi Preface E N Asia Pacific Amersham Pharmacia Biotech Asia Pacific Ltd 15 F North Cornwall H ouse 979 King s Road Taikoo Place Quarry Bay Hong Kong Tel 852 2811 8693 Fax 852 2811 5251 Web site www apbiotech com applications microarray xii Lucidea Microarray ScoreCard User s Guide Caution Chapter 1 Introduction Lucidea M icroarray ScoreCard v1 1 consists of the control plate and the software This chapter provides an overview of the control plate and the software The topics are e About the Lucidea M icroarray ScoreCard control plate section 1 1 e About the Lucidea M icroarray ScoreCard software section 1 2 e Workflow overview section 1 3 e Example files on the Lucidea M icroarray ScoreCard CD section 1 4 1 1 About the Lucidea Microarray ScoreCard control plate In evaluating microarray experiment results you must first minimize variations in the measurements so that accurate comparisons can be made within an experiment and across multiple experiments O ne method of minimizing the variations is the use of control samples so that you can relate the experiment data to the control data in a quantitative manner You use the control plate in your experiments for this purpose The control plate is a 384 well microplate that contains 12 replicates of 32 control samples For more information about the control samples see chapter 2 W hen using the M icroarray System you can set
60. res Pf EPS ee A Le Bae D i vI ET T wae Le D ar T P i Er ae Dale Ee ee ae ee ee ee a Figure 8 9 The normalized data file in Excel Because of its size only a small portion of the report is shown Included with the image analysis output measures are e Detection limit flags lt C y3 DL lt Cy5 DL The flags indicate whether the signals fall below the detection limit A 1 indicates the signal is below the detection limit A 0 indicates the signal is above the detection limit e Spot ULR The uncorrected log ratio for the particular spot e Spot NLR Thenormalized log ratio for the particular spot e Norm Cy3 The normalized Cy3 signal for the particular spot which is Cy5 signal 10 SPot NLR e ULR 1 Theuncorrected log ratio for the particular spot in spot set 1 e ULR 2 The uncorrected log ratio for the particular spot replicate in spot set 2 Lucidea Microarray ScoreCard User s Guide p8 13 Chapter 8 Viewing the analysis results SSS e NLR 1 The normalized log ratio for the particular spot in spot set 1 e NLR 2 The normalized log ratio for the particular spot replicate in spot set 2 For an explanation of how these values are calculated see appendix A The normalized data file structure matches the requirements for Spotfire Pro and ArrayStat Statistical Informatics software You can open this file directly in Spotfire Pro to display the Cy5 versus normalized Cy3 scat
61. s section A 2 3 e Adjusting the normalized log ratios section A 2 4 e Verifying the normalized results section A 2 5 A 2 1 Working with the data in logarithmic scale The software works with the data in logarithmic scale log base 10 because the distribution of the data becomes normal symmetrically centered about x 0 when the genes are not differentially expressed in logarithmic space This transformation facilitates the statistical analysis of the data Table A 1 demonstrates the differences between the untransformed and log transformed ratios of the Cy3 to Cy5 signals The untransformed ratios show that the corresponding pairs of up and down regulated genes ratios do not center about 1 the ratio of the genes when they are not differentially expressed After the log transformation all the corresponding pairs of up and down regulated ratios are distributed symmetrically about 0 the log of the ratio of the non differentially expressed genes Table A 1 Transforming ratios into logarithmic scale Gene Ratio Logo Ratio A not differentially expressed 1 0 00 B 2 fold up regulated 2 0 30 C 2 fold down regulated 0 50 0 30 D 4 fold up regulated 4 0 60 E 4 fold down regulated 0 25 0 60 F 10 fold up regulated 10 1 00 G 10 fold down regulated 0 10 1 00 pA 2 Lucidea Microarray ScoreCard User s Guide A 2 Normalizing the data A 2 2 Calculating the uncorrected log ratio For each spot on the slid
62. shold for the mean NLR SD section A 4 N ote This appendix assumes that you are familiar with statistical and mathematical terms and concepts commonly used in scientific experiments A 1 About the normalization method Normalizing microarray data is an essential first step in the correct interpretation of the gene expression experiment results For two color experiments the Lucidea M icroarray ScoreC ard uses the exponential normalization method to correct for artifacts in studies using Cy3 and Cy5 dyes Experiments show that e Therequired normalization factor is not constant but is a function of the Cy5 signal e The magnitude of the artifact varies among all the hybridized slides Therefore the normalization equation parameters must be determined empirically for each slide The exponential normalization method is advantageous because it is based on all the data not just a single control gene or a series of external spikes The method improves normalization particularly for weakly expressed genes and results in better centering and tighter distribution for the non differentially expressed genes Lucidea Microarray ScoreCard User s Guide pA 1 Appendix A Data normalization and statistical significance S A 2 Normalizing the data This section describes the following e Working with the data in logarithmic scale section A 2 1 e Calculating the uncorrected log ratio section A 2 2 e Calculating the normalized log ratio
63. sion make sure you e Choose the N ormal spotting mode in the Generation III Array Spotter Control software N ote For system verification only you can use the Single Plate spotting modein Array Spotter Control to spot one control plate repeatedly e Choose the N ormal spotting mode or the High Density spotting mode Duplicate spotting pattern in the ASC XT software Note M ake sure you set the spotting parameters so that all the control elements from each pen are spotted in sequence e Select the Complete Last Row option in the ASC XT software so that each row contains an equal number of spots e Use only full microplates 32 well sets Using partial microplates causes calculation errors in the software For detailed instructions on how to set the spotting parameters see the M icroarray System Generation 111 User s Guide If you are using the ASC XT software see the Microarray System ASC XT User s Guide for the Generation III Array Spotter After you set up the Array Spotter you can begin the spotting session p2 6 Lucidea Microarray ScoreCard User s Guide Important 2 6 Setting up the microscanner 2 6 Setting up the microscanner To scan the slides containing the spotted control samples you use the typical procedure for your microscanner If you plan to use reflective slides from Amersham Pharmacia Biotech see appendix B for instructions on how to adjust the PMT setting to reduce pixel saturation and eliminate in
64. software related instructions in this guide assume that you have basic computer skills You should be familiar with the M icrosoft Windows NT or Windows 2000 graphical user interface If you do not have these skills consult the appropriate documentation or refer to the H elp file for the operating system x Lucidea Microarray ScoreCard User s Guide Assistance Assistance If you have questions about or require assistance with the Lucidea M icroarray ScoreC ard control plate or the Lucidea M icroarray ScoreC ard software contact your local Amersham Pharmacia Biotech service office For the local office nearest you see the Amersham Pharmacia Web site You can also contact one of the following offices US and Canada M olecular Dynamics Inc 928 East Arques Avenue Sunnyvale CA 94085 4520 USA Tel 1 800 743 7782 or 1 408 773 1222 Fax 1 408 773 1493 Email masupport am apbiotech com International Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England H P7 9N A Tel 44 1494 544000 Fax 44 1494 542266 Europe Amersham Pharmacia Biotech Europe GmbH M unzinger Strasse 9 D 79111 Freiburg Germany Tel 49 76 1490 30 Fax 49 76 1490 3159 Japan Amersham Pharmacia Biotech KK Otsuka Daiichi Seimei Bldg 32 22 Higashi Ikebukuro 2 chome Toshima ku Tokyo 170 Japan Tel 81 3 5331 9317 Fax 81 3 5331 9372 Lucidea Microarray ScoreCa
65. t Expression e Inthe Arrays area select 4 from the N umber of arrays list 5 4 Loading the images From the navigation pane choose Images under Analysis The Images pane appears figure 5 2 In the Images pane retrieve the four image files in the following order e L1 Spot set 1 left channel 1 e L2 Spot set 1 left channel 2 e R1 Spot set 2 right channel 1 e R2 Spot set 2 right channel 2 Protocol Editor x Protocol Copy of md 1 Overview ca e rs E Analysis Images Name File El Layout 1 Data Data 1 000006347_L_1 GEL Blanks 2 Data Data 2 000006347_L_2 GEL Display lt a on ata S AEL Labels 4 Data 4 EL E Measures Background References E Alignment Anchors Post analysis gt I Use image filename as channel name Correct image offset error File Refresh Cancel Help Figure 5 2 Loading images in ArrayVision 5 1 Lucidea Microarray ScoreCard User s Guide p5 3 Chapter 5 Preparing ArrayVision 5 1 analysis files for import SSS 1 5 5 Setting up the layout of the array Use the guidelines in this section when you set up the array layout for analysis From the navigation pane choose Layout The Layout pane appears figure 5 3 Protocol Copy of md 1 m Template organization Overview R C Level 3 Sub arrays E Analysis a M Level 2 Spot groups Images ae v Level 1 Spots
66. ter plots and visualize the spot set 1 and spot set 2 comparisons You can open this file directly in ArrayStat Statistical Informatics to identify outliers and determine which genes are significantly differentially expressed p8 14 Lucidea Microarray ScoreCard User s Guide Chapter 9 Comparing data from multiple slides This chapter explains how to assess the M icroarray ScoreCard results and compare the data from multiple slides The topics in this chapter are e Assessing data quality and statistical significance section 9 1 e Interpreting the Gene Expression data from single and multiple hybridizations section 9 2 9 1 Assessing data quality and statistical significance Before you evaluate M icroarray experimental results you should use a common set of criteria across a batch of slides to accept or reject a slide in the experiment The criteria are based on the quality measures that the Microarray ScoreCard software calculates The software uses the thresholds you set to determine whether or not the quality measurements are within the acceptable range This section provides some guidelines you can use to accept or reject the slides when you evaluate the M icroarray ScoreCard results The quality attributes that should be taken into consideration and the corresponding quality measures calculated by the software are discussed For information about how to use the software see the H elp available in the software For a descri
67. that the average NLR SD will typically increase indicating a better correlation between the normalization equation with the Cy5 signals than with the Cy3 signals e You can usetheinformation in the normalized data file section 8 9 to plot and view the relationship of the following Thenormalized log ratio for each spot Spot NLR and the log of the Cy5 signals log Cy5 The normalized log ratio of each spot Spot NLR and the log of the Cy3 signals log Cy3 The plots should show that the Spot NLR values center around x 0 with no significant relationship between the signal level and NLR Alternatively you can plot the normalized Cy3 signal values N orm Cy3 against the normalized Cy5 signal values Norm Cy5 The plot should form a line with a slope of 1 0 A 3 Estimatingtheconfidenceintervals forthe gene expression data The software calculates the average standard deviation of the normalized log ratio for each control element from 24 replicates Experiments have shown that for most hybridizations the standard deviation for each grouping is approximately the same for those groups whose signal is above the detection limit Therefore the software calculates and reports the M ean NLR SD as an approximation of the variation in gene expression ratios across the _ hybridizations pA 4 Lucidea Microarray ScoreCard User s Guide A 4 Calculating the default threshold for the mean NLR SD In statistical analysis you c
68. the analysis results from both versions are almost identical pl 4 Lucidea Microarray ScoreCard User s Guide Chapter 2 The Lucidea Microarray ScoreCard control reagents This chapter describes the control samples in the Lucidea M icroarray ScoreCard control plate how to use the control samples and the layout of the control samples on a slide The topics in this chapter are e Control samples and functions section 2 1 Spike mix composition and use section 2 2 Storing the control plate section 2 3 e Preparing the control plate for spotting section 2 4 e Setting up the Generation III Array Spotter section 2 5 e Setting up the microscanner section 2 6 e Control element positions on a slide section 2 7 Note Currently the control plate requires and is limited to two color analysis 2 1 Control samples and functions Table 2 1 lists the control samples their positions in the control plate and the corresponding identification in the software For detailed instructions on how to prepare the control samples for spotting see the instructions shipped with the control reagents Lucidea Microarray ScoreCard User s Guide p2 1 Chapter 2 The Lucidea Microarray ScoreCard control reagents E Table 2 1 Control samples and positions in a microplate ID in Well set well positions Control sample type Description ScoreCard 1 A1 throu
69. the analysis results chapters 3 through 6 Import the image analysis data file s for one slide into the Lucidea Microarray ScoreCard software and process the data See chapter 7 and the software Help for instructions 1 View and interpret the processed data chapter 8 Figure 1 1 Lucidea Microarray ScoreCard workflow Lucidea Microarray ScoreCard User s Guide p1 3 Chapter 1 Introduction 1 4 Example files on the Lucidea Microarray ScoreCard CD In addition to the software and documentation the Lucidea M icroarray ScoreCard CD contains the following example files in the M icroarray folder Control_elements_image tif An image file that shows three pen areas Each pen area consists of three rows or three microplates of control elements Lucidea Automated Spotfinder 1 0_Ir_1 2 txt A set of four text files containing the output from Lucidea Automated Spotfinder 1 0 415 ArrayVision 4 0 txt A text file containing the output from ArrayVision 4 0 using the required setup as described in chapter 3 415 ArrayVision 5 1 txt A text file containing the output from ArrayVision 5 1 using the required setup as described in chapter 4 GenePix Pro 3 0 gpr A GenePix Pro results file containing the output from GenePix Pro 3 0 Control_Plate_content txt A text file containing clone information N ote T he same image file was loaded into ArrayVision 4 0 and 5 1 so
70. thods except the following Selected spots and Selected spot groups Lucidea Microarray ScoreCard User s Guide p5 7 Chapter 5 Preparing ArrayVision 5 1 analysis files for import Protocol Editor x Protocol Copy of md 1 Dvaiview WV Enable background subtraction Analysis Images Layout Blanks Display E Labels ispl i a Ga Defined distance 2 pixels thickness 2 pixels Background References p Mode Alignment Individual C Average Surrounding entire template Surrounding spots Comers between spots Surrounding spot groups Selected spots pan oe ET OY oj Anchors Post analysis File Refresh Cancel Help Figure 5 8 The Background pane 5 9 Defining the references optional A reference is the calculated value to which all array elements are normalized You do not need to define a reference because Lucidea M icroarray ScoreC ard will normalize the data for you H owever if you choose to enable reference use select only one of the following All spots User defined areas 5 10 Analyzing the image files After you finish setting up the parameters as described in this chapter analyze the image files using your usual process 5 11 Exporting the analysis data After ArrayVision finishes the analysis export the analysis data to tab delimited text file s These files can be imported into the Lucidea M icroarray
71. tical analyses involving the normality test of residuals and the identification and removal of outliers pA 6 Lucidea Microarray ScoreCard User s Guide Caution Appendix BUsing the Amersham Pharmacia Biotech reflective slides with Lucidea Microarray ScoreCard This appendix explains the effects of reflective slides on data normalization and how to adjust your slide scanning techniques to produce usable data for analysis The topics in this appendix are e Effects of the reflective slide on data normalization section B 1 e Detecting the saturated pixels section B 2 e Eliminating the saturated pixels section B 3 B 1 Effects of the reflective slide on data normalization The reflective slide technology from Amersham Pharmacia Biotech increases the signal strength and sensitivity in microarray experiments Although these slides allow you to detect very weak signals spots with extremely high signals can become saturated in the image For the Generation III Array Scanner saturated pixels are pixels that reach 100 000 rfu the maximum signal measurement Because the true signal levels are underestimated saturated pixels result in inaccurate spot quantitation This phenomenon impacts the validity of the data normalization results regardless of the method used The greater the number of saturated spots the larger the impact on data normalization Quantitation results will notbeaccurate foranyspotthatcontains saturated pixels
72. ts in red all Pen Variation results with absolute values exceeding the threshold you set The default threshold setting is 25 For example if you use the default threshold for pen variability all the results with absolute values exceeding the 25 threshold are highlighted in red figure 8 7 The highlights indicate possible problems and should be considered when you evaluate the data Negative Pen Variation values indicate pen areas with mean signal below the overall mean housekeeping gene values for the entire slide A large percent variation that occurs consistently in the same pen across multiple slides from the same spotting session can indicate a problem with that pen Relatively large percent pen variation values may indicate problems with pen performance However process steps after spotting can also contribute to the variation Therefore you should take other factors into consideration when reviewing the large percent variation 8 7 2 Spot set to spot set variability The second table in the System Validation window figure 8 7 shows spot set to spot set variability using the following data e Mean For each spot set the average of all Cy3 or Cy5 signal values from the actin gene in well sets 14 and 29 CV The corresponding percent coefficient of variation for each average value e Spot Set Variation Percent variation of the signal from the spot set Mean Spot Set Meanactin Meanactin 100 M ea
73. up your experiment to include the control plate The control samples are placed on each slide to allow spot to spot comparisons within and across multiple slides 1 2 About the Lucidea Microarray ScoreCard software The software must be used only with the control plate Using the software with other control samples will not provide accurate results The software processes microarray experiment data from one slide Using the results from the software you can compare the data from one slide with the results from other slides within and across multiple experiments Lucidea Microarray ScoreCard User s Guide pl 1 Chapter 1 Introduction Saas The software e Allows you to import two color image analysis data from Lucidea Automated Spotfinder ArrayVision or GenePix Pro e Processes the experiment data from one slide by calculating the quality measures e Normalizes the data on the slide e Correlates the sample information to the experiment values e Performs additional calculations so that you can assess the system performance e Prints and exports all the processed results to tab delimited text files Important The software does not provide an absolute scale for the assessment of the experiment data You must accept or reject the data based on your interpretation of the calculated results The software requires e 233 MHzor faster Pentium or equivalent computer 64 MB RAM or more Windows NT 4 0 Service Pack 5 or better
74. xample analysis file generated by ArrayVision 4 0 is in the M olecular D ynamics M icroarray M icroarray ScoreCard folder 4 1 The image and analysis data files In two color microarray experiments the Generation III Array Scanner produces four image files in the following order e Spot set 1 left dye 1 e Spot set 1 left dye 2 e Spot set 2 right dye 1 e Spot set 2 right dye 2 Lucidea Microarray ScoreCard User s Guide p4 1 Chapter 4 Preparing ArrayVision 4 x analysis files for import SaaS When you perform image analysis in ArrayVision you can produce one of the following e A single analysis file containing data from all four image files e A set of four individual analysis files each containing the data from one of the four image files compatible with Gen3DB The Lucidea M icroarray ScoreC ard software can analyze the data in either file type H owever the software processes only files with specific field names and the fields must bein a specific order Therefore you must make sure that the analysis itself is set up properly for producing the correct output file s 4 2 Setting the display format In ArrayVision how you display the images in the window affects how the image analysis data is produced a single file that contains data from all four images or four individual files each containing data from a single image When you set up the display format be sure to use one of the following settings e

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