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1. unknown or GEX If you would like to make changes to this file please be sure that when you save the changes to the text document you maintain the file structure Spacing columns etc Save the file as txt or csv Assay Name Type hsa miR 548d Target EXAMPLE hsa miR 123 Target U6 snRNA endogenous control n2343 Candidate Control SOFTWARE TIPS Sample Design Table e To copy a group assignment to all samples click on that group name then right click and select Copy To All This will assign that group to all samples Choosing Clear All will delete all group assignments from all samples e To omit one or more samples from analysis highlight one or more samples then right click and select Omit Sample from Study You may also check the Omit box to the right of the Group e Select two or more samples to view this subset of samples only in the results plots Page 10 of 13 Assay Design Table To copy an assay type assignment to all assays click on that assay type then right click and select Copy To All To omit one or more assays from analysis highlight one or more assays then right click and select Omit Assay from Study You may also check the Omit box to the right of the Type To view a subset of assays select two or more assays then only those assays will be shown in the results plots C Data tab This table contains information imported from the real time data results files imported file path well
2. 0 8918 0 6768 0 9542 2 oa 1 8039 1 0409 1 2875 1 0971 1 3555 1 2561 1 3496 1 4062 0 8989 1 4219 LHA L2873 1 2436 12229 1 3084 0 9942 1 7668 1 1709 1 3387 12169 1 0729 1 0789 1 0796 1 0905 1 0981 1 1015 1 1119 L115 1 1134 1 1169 1 1138 0 985 0 9745 0 8693 1 0029 0 9578 0 986 0 9458 0 9779 0 349 1 027 0 9885 1 0298 1 0258 1 0083 0 9625 0 913 1 0537 1 0284 10259 10125 1 0915 1 0867 rae 1 0371 10751 0 9385 L351 L05381 0 3041 L2457 1 5465 1 1531 1 024 0 9245 0 9304 0 8187 L139 0 9632 0 7624 1 0364 1 3506 1 0267 0 886 1 3857 1 0625 0 7444 L151 0 8555 0 8531 0 8421 0 8611 0 8558 0 8846 1 0755 1 0759 Lolz 1 1624 10721 1 4974 1 7708 L25 1 184 1 9716 1 3255 1 1366 LHL L1127 LUA 1 0167 1 2537 Reference ma a mamn mea et ad ee L RO Max 1 0 1 0 Brain MM1 g 1 eae Got 0 9351 0 9521 0 7056 0 8905 0 9828 0 9592 0 8665 0 9667 0 985 0 9766 0 7266 0 907 0 9684 0 8618 0 8656 0 9163 0 9526 0 9857 0 7862 0 8978 0 9027 0 9328 T 2 1 0503 L4173 L123 1 0175 L25 LiH L035 1 0153 1 024 L363 1 1025 1 0326 1 1603 1 1552 i 1 0913 1 048 1 0145 L272 1 1138 1 1078 L072 QC Plots help to visualize sample and group correlations for a quick quality check of data Graphic Results Plots are helpful to visualize your analyzed data All plots have a mouse over functionality to provi
3. Cr Data The Cr Data tab displays a table with the following information from the real time data files imported file path well number assay name assay type sample name group name C value Adjusted Cy value and reason for omission of sample well from calculation Changes made to Sample or Assay Design information will be reflected in the C Data tab only after performing analysis Experiment Design CT Data Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings
4. File gt Export In the Export dialog box that is opened you may select individual files csv or txt or one file xls with each table in a separate worksheet to be exported Tables that can be exported are the C Data Sample Design Assay Design Average Cy ACz 2 and Fold Change files Open a Saved Study Open a study you have previously saved by either clicking on the S icon in tool box or File gt Open Browse to find the file containing the results data from the desired study the das file containing the analyzed data and select to open Experiment Data Experiment Design Sample Design e Sample Name The name given to each sample by the user is pre defined in and imported with the results files Data points in a study with the same sample and assay name are considered technical replicates e Group The name of the biological replicate group e g normal disease or time point 1 time point 2 o Enter or import Sample Design information To assign samples to biological groups such as normal amp disease or time point 1 time point 2 either click in the Group box and manually enter group name or upload a sample design file see Appendix A Click on Browse to find and select your sample design file If your samples are solely technical replicates not biological replicates no group assignment is necessary Note Samples labeled as NTC no template control are not used in calculations Page 3 of 13
5. or Single Linkage Map Type Assay Centric Global View default or Sample Centric For each Map Type the AC value of the neutral middle expression level mean or median is set such that red indicates an increase with a AC value below the middle level and green or blue indicates a decrease with a AC value above the middle level Global View The middle expression level is set as the median of all the AC values in the study by default and can be adjusted using the scale on the right side of the plot The scale for the plot can be changed between the AC and the AC Plus global control mean or global median Any data point in the plot can be compared relative to all others Assay Centric For each assay the middle expression level is set as the median of all of the AC values from all samples for that assay Data points for a given assay can only be compared relative to other data points for that assay Sample Centric For each sample the middle expression level is set as the median of all of the AC values from all assays for that sample Data points for a given sample can only be compared relative to other data points for that sample loxi Distance Measure Pearson s Correlation Clustering Method Average Linkage MapType CCCEAGIE Color Scheme Red Green Heat Map Study iu a RELA H300153294_m1 TNFRSF21 H300205419_m1 CASP Hs00169152_m1 BCL2L13 Hs00209789_m1 BIRC5 Hs00977611_91 BCAP31
6. outliers represented in a box plot are not the same as outliers calculated by the modified Grubb s calculation that is used to exclude outliers from the data for further calculation This plot is useful for viewing the variation in the Cry values among biological replicates or assays Signal correlation Displays C signal correlation between samples in a chosen Group Pearson s product moment correlation coefficient r is calculated for each pair of samples in the selected Group and plotted on the Signal Correlation Plot This plot can be displayed in a Red Green or Red Blue color scheme Note Signal correlation plot is for biological replicates and is not drawn if no Group is entered in Sample Design table aasa Ei DR Ca a P pily T a lalz Page 7 of 13 Scatter Plot Displays AC correlation between samples within a chosen Group Pearson s product moment correlation coefficient r is calculated for each pair of samples in the selected Group and plotted on the Scatter Plot respectively Graphic Results Plots RQ Plot Displays RQ log fold change vs Target or RQ vs Sample The Graph Types available to view the data are Linear Logi9 and Logp If no Group is specified the standard deviation of the AC is also plotted for each sample on the Logz Graph Type Volcano plot Displays P values vs Fold Change of Groups based on input Fold Change Boundary and P values Default is a Fold Cha
7. the C Data tab as outlier replicate e Adjust p values using Benjamin Hochberg False Discovery Rate FDR If you choose this option multiple testing corrections will be done to adjust p values to correct for occurrence of false positives using the Benjamini Hochberg False Discovery Rate method If you have selected the option to use the FDR method then all displayed p values will be the adjusted p values e Choose Normalization Method This allows for two different methods of normalization use of one or more endogenous control s or global normalization Mestdagh et al 2009 When more than one endogenous control gene is selected for normalization the software calculates the mean of the chosen endogenous control genes to use as a normalizer normalization factor on a per sample basis Global normalization first finds the common assays among all samples The median Cy of those assays is used as the normalizer on a per sample basis e Select Endogenous Controls For Analysis Click on the binoculars icon to see a plot displaying C values of all samples for all assays that are labeled as Candidate Control or Selected Control in the Assay Design table The box to the right lists each candidate selected control gene and the Score value based on standard deviation see Appendix B calculated for those assays The lower the score the more stable the control You may select one or more assays to use as an endogenous control s e Reference sa
8. DataAssist v3 0 Software User Instructions Introduction DataAssist Software is a simple yet powerful data analysis tool for sample comparison when using the comparative Cr AAC method Livak and Schmittgen 2008 for calculating relative quantitation of gene expression It contains a filtering procedure for outlier removal various normalization methods based on single or multiple genes and provides relative quantification analysis of gene expression through a combination of statistical analysis and interactive visualization What DataAssist Software does The main steps in the analysis performed by DataAssist Software are e Read export files txt or csv of analyzed results from supported instruments and software e Perform QC analysis on Cr data and associated plots e Perform sample normalization for each assay e Perform QC analysis on normalized data e Perform relative quantification for sample comparison perform t test for biological group comparisons and produce graphics to visualize test results Required Files and Formats DataAssist Software is designed to work with txt or csv results files exported from Applied Biosystems Real Time PCR instruments and is able to process multiple Relative Quantitation RQ study files simultaneously Thus very large studies across a large number of plates or microfluidic cards can be analyzed as one data set DataAssist Software is compatible with analyzed real time res
9. Hs01036137_m1 PYCARD Hs00203118_m1 BNIP3L Hs00188949_m1 NFKBIZ Hs00230071_m1 PMAIP1 Hs00560402_m1 TNF H00174128_m1 LTA Hs99999086_m1 BCL2L10 Hs00368095_m1 BCL2L14 H900373302_m1 oix Clustering Method Average Linkage Y Map Type Assay Centric Color Scheme Red Green Distance Measure Pearson s Correlation Y Heat Map Study iu a ee m Mm RELA H300153294_m41 TNFRSF21 H300205419_m1 CASP Hs00169152_m1 BCL2L13 Hs00209789_m1 BIRC5 Hs00977611_91 BCAP31 Hs01036137_m1 PYCARD Hs0203118_m1 BNIP3L Hs00188949_m1 NFKBIZ Hs00230071_m1 PMAIP1 Hs00560402_m1 TNF H00174128_m41 LTA Hs99999086_m41 BCL2L10 Hs00368095_m41 BCL2L14 Hs00373302_m41 lolx Distance Measure Pearson s Correlation Y Clustering Method Average Linkage Y Map Type Color Scheme Red Green Heat Map Study iu re RELA H300153294_m41 TNFRSF21 Hs00205419_m41 CASP Hs00169152_m1 BCL2L13 Hs00209789_m1 BIRC5 Hs00977611_91 BCAP31 Hs01036137_m1 PYCARD Hs00203118_m1 BNIPSL Hs00188949_m1 NFKBIZ H30023007 1_m1 PMAIP1 Hs00560402_m41 TNF Hs00174128_m1 LTA Hs99999086_m1 BCL2L10 Hs0368095_m1 BCL2L14 H90373302_m1 h All plots shown here are shown with Pearson s Correlation and Average Linkage Map types are shown in this order Global View Assay Centric then Sample Centric Page 9 of 13 Appendix A Experimental Design Files Experimental Design files can be used to import sample and assay information Start with eithe
10. de quick access to sample information Right clicking on any plot gives you the option to Copy Save as or Print the figure Note To view only a subset of assays in any of the Graphic Results Plots select two or more assays Then only those assays will be shown in the results plots Page 6 of 13 Endogenous Control Selection Displays C values of candidate and selected controls for all samples as well as a calculated score see Appendix B Checking the box to the right of an assay will assign it as a selected control One or more controls may be selected for data normalization If you choose more than one gene for normalization the mean C value of the controls will be used for normalization Note A minimum of 2 controls are needed to calculate the score Since the score is relative to other controls the score will be the same if you only have two controls QC Plots Box Plot Displays the overall range of C distribution shown by Sample sorted and colored by Group or by Assay The box contains the middle 50 of the data Cr values The black horizontal line and the black dot denote the median C value and mean Cr respectively The ends of the vertical lines whiskers indicate the minimum and maximum C values unless these values exceed 1 5 x IQR The IQR is the inter quartile range the distance between Q1 and Q3 The points outside the ends of the whiskers are outliers or suspected outliers Note The
11. hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Documents and Settings hegeripa Desktop For examples Assay Design Liver2 Liver2 Liver1 Liver 1 Liver2 Liver2 Liver1 Liver 1 Liver2 Liver2 Kidney 1 Kidney 1 Liver2 Liver2 Liver 1 Liver 1 Brain1 Brain1 Kidney2 Kidney2 27 8615 27 8603 31 9428 31 8011 31 7115 31 9237 27 9246 27 8492 25 1618 25 1653 25 1341 25 1602 29 5247 29 5947 25 1899 25 0787 26 7171 26 7747 25 2224 25 2159 27 8615 27 8603 31 9428 31 8011 31 7115 31 9237 27 9246 27 8492 25 1618 25 1653 25 1341 25 1602 29 5247 29 5947 25 1899 25 0787 26 7171 26 7747 25 2224 25 2159 Dataassist Software v1 999 ee 18 x File Help Ooo a prev ore Experiment Data Assay Name The detector or target name assigned by user imported in with the results files This is most often the Assay ID gene name or the name of the assay provided by the user Assay Type The task assigned by user if required e g target endogenous control imported in with the results files Assay Design File Assay Design information can be entered by using an assay design file see Appendix A Alternatively you can enter this information into the analysis software using the drop down menu provided Enter or import Assay Design information To assign assay types Target Selected Control or Candidate Control either click in the T
12. icient r is calculated considering all AC values from all assays and the distance is defined as 1 r For an assay pair the r is calculated considering all AC values from all samples and the distance is defined as 1 r e Euclidean Distance sqrt gt AC i AC j 42 For a sample pair the calculation is done across all assays for sample i and sample j For an assay pair the calculation is done across all samples for assay i and assay Bibliography Livak K J and Schmittgen T D Analyzing real time PCR data by the comparative CT method Nature Protocols 3 1101 1108 2008 Mestdagh P Van Vlierberghe P De Weer A Muth D Westermann F Spelemean F Vandesompele J A novel and universal method for microRNA RT qPCR data normalization Genome Biology 10 R64 2009 Vandesompele J De Preter K Pattyn F Poppe B Van Roy N De Paepe A Speleman F Accurate normalization of real time quantitative RT PCR data by geometric averaging of multiple internal control genes Genome Biology 3 research0034 2002 For Research Use Only Not intended for any animal or human therapeutic or diagnostic use 2011 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Page 13 of 13
13. mple group calibrator This is the control sample or group to which you want to compare your other samples or groups When running the t test this makes up control group against which the test group is compared e Perform Analysis Click to start the analysis Page 5 of 13 Analysis Results e Average C Average C value of replicates e ACz Normalized C values standard deviation only applied to technical replicates Q AcT Changes AC values to linear values e Fold Change RQ Displays fold change RQ RQ Min and RQ Max for each sample For biological groups fold change and P value will be displayed Analysis Results Average Cr ACT 28Cr Fold Change RQ a ee Target Target sa miR 135a Target samiR 532 5p Target sa miR 383 Target sa MiR 34a Target samiR 195 Target sa miR 124 Target sa miR 106b Target sa miR 26b Target sa miR 339 3p Target sa miR 127 3p Target hsa miR 142 3p Target sa MiR 331 3p Target sank 886 3p Target sa miF 382 Target sa mik 92a Target sa miF 28 3p Target sa miR 20b Target sa miR 99b Target sa miR 150 Target sa miR 191 Target QC Plots and Graphic Results Plots 1 074 1 1702 L231 0 9219 L1435 0 9518 1 189 1155 L079 11525 0 7968 1 2352 L271 11398 0 9134 1 0835 11625 0 8813 1 1649 1 0219 0 9518 1 0775 eee 0 8429 0 8166 1 0153 0 8257 1 043 0 9907 0 8627 0 9446 0 7063 1 073 1 0417 1 0092 0 6708 1 0329 0 7813 0 7681
14. nge Boundary of 2 2 fold change and a P value of 0 05 Note Volcano plot is not drawn if no Group is entered in Sample Design table as no p values are calculated in this instance Bij Fist Beiren E eree a See cates ft Pie m E 02 E on i Halah ey my Page 8 of 13 Cluster Analysis amp Heat Map Graphically displays results of unsupervised hierarchical clustering Distances between samples and assays are calculated for hierarchical clustering based on the AC values using either Pearson s Correlation or Euclidean Distance This plot can be displayed in the Red Green or Red Blue color scheme and has a zoom in zoom out feature on the left of the plot Sample names are colored by group if groups have been designated Both AC and AC global control mean global median if global normalization was used for normalization are displayed in the mouse over tool tip The global contro mean is the mean C value of all selected endogenous controls in the study The global median is the median value used if global normalization was use This value global control mean or global median is added on to the AC to better approximate the original Cy a rough estimate of expression level calculated for each sample and given assay prior to normalization Distance Measure Pearson s Correlation default or Euclidean Distance Clustering Method Average Linkage default Complete Linkage
15. number assay name sample name and C value Adjusted C will contain a different C value only if you have set the Maximum allowable Cy value to lower than the last cycle of your real time run i e lower than C 40 Omit column contains information if well has been omitted QC Plots and Graphic Results To zoom in click and drag the mouse over desired region in the plot To zoom out click and drag the mouse up To update the Volcano Plot graphic after changing the P value or Fold Change Boundary hit enter once you ve entered the desired number Mouse over data points to get sample related information such as sample name assay name AAC values P values etc Right click in plots to Copy figure Save As png or jpg image files or Print figure To view only a subset of data in any of the Graphic Results Plots select two or more assays from the Assay Design table or two or more samples from the Sample Design table Select the Results plot button and only that subset of assays and or samples will be displayed in the results plots From the Analysis Results table you may sort the data and select a subset of assays to have plotted on the Heat Map RQ Plot or Volcano Plot Appendix B Analysis Workflow and Calculations in DataAssist Software 1 The C values for each well are adjusted and included excluded for analysis based on the following analysis settings Maximum allowable C value Max Cr if a C 2 Max Cy it is adju
16. r Excel or WordPad or equivalent to create a design file If using Excel save the files as a txt or csv file Design files must have 2 columns and it is important to maintain the file structure spacing columns Sample Design File The sample design file should have two data columns Sample Name and Group separated by a comma or a tab The rows of this file represent the samples used in an experiment The first column must be named Sample Name or SampleName and it will contain the same sample names used in the real time PCR results files The second column must be named Group or Type Use the Group column to designate biological groups i e treated and untreated Enter in the groups you would like to be identified Save the file as txt or CSV Sample Name Group ABC123 untreated ABC456 untreated EXAMPLE ABC789 untreated DEF123 treated DEF456 treated DEF 89 treated Assay Design File The assay design file should have two data columns Assay Name or assayName and Type separated by a comma or a tab the Type column can be Target GEX Candidate Control Selected Control or Endogenous control The rows of this file represent the assays genes The first column is Assay Name and will contain the same names as your detectors or targets from the SDS output The second column is Type and will contain the task information real time PCR results i e target endogenous control
17. s are specified for each sample group 24 of the samples in the group ACt o Calculate the geometric mean of ACtreference o Fold Change RQ geometric mean 2 geometric mean 2 o A two sample two tailed Student s t test comparing the AC values of the two groups is performed and a p value is calculated if both groups have 2 or more samples The p value is adjusted if the analysis setting Adjust p values using Benjamini Hochberg False Discovery Rate is checked Benjamini Hochberg FDR method is based on R package multtest at http cran r project org web packages multtest index html If no sample group is specified for each sample o RQ 2 ACt g ACtreference o Standard deviation SD is calculated for C values of the technical replicates and is used to calculate the RQ Min and RQ Max Page 12 of 13 RQ Min JACt SD a BCtreterence RQ Max 2 ACt SD l JMACIererenge 6 Pearson s product moment correlation coefficient r is calculated for C or AC values of sample pairs and plotted on the Signal Correlation Plot and Scatter Plot respectively NXXY ZXXZY NZX SXP VND ZYY 7 Unsupervised hierarchical clustering is performed and then displayed as a Heat Map Distances between samples and assays are calculated for hierarchical clustering based on the AC values using one of the following e Pearson s Correlation For a sample pair the Pearson s product moment correlation coeff
18. says is used as the normalizer on a per sample basis 4 Select one or more Endogenous Controls for analysis The score of each candidate or selected control is the average pairwise variation of that gene with all other chosen candidate or selected control genes It is calculated as shown below Vandesompele et al 2002 Note The score is only calculated once There is no iterative exclusion of the highest value If you would like to exclude the gene with the highest value and recalculate you may change the assay type of that gene from candidate control to target and the software will recalculate the stability value for the remaining genes For control i calculate AC for all samples using another control j as the normalizer and calculate the standard deviation SDj of the AC values Repeat the above SDij calculation for all other candidate controls j 1 N 1 and use the average of all SDij s as the stability score for control i Note A minimum of 2 controls are needed to calculate a score Since the score is relative to other controls it will be the same if you only have two controls 5 Once controls are selected click the Perform Analysis button The results are calculated as following For each sample o Average C mean of the technical replicates o AC Average C Normalization Factor NF endogenous control o Act O is also calculated for determining Fold Change If sample groups biological replicate
19. sted to Max Cr The undetermined Cr is also converted to Max Cr Include max C values in calculations If Yes is selected wells with Max C are included in the analysis If No is selected any well with Max C is excluded from analysis Note Go to the C Data tab to see any changes in both the Adjusted C and Omit columns If analysis setting Exclude outliers among replicates is checked a refined Grubbs outlier test is applied together with a heuristic rule to remove the outlier among technical replicates Find the replicate whose C value has the largest absolute deviation from the mean C value and calculate the deviation G in units of the standard deviation SD G max C mean C SD Page 11 of 13 If the following test is true and max C mean C7 2 0 25 then the replicate with max Cy is removed as outlier o gt WH _ Aevama n VN N 2 8 ama 2 Fa f 2 N 2 is the critical value of the t distribution with N 2 degrees of freedom and a significance level of a 2N a 0 05 is used Note Go to the C Data tab to see outliers in the Omit column 3 Chose normalization method Endogenous Control Choose one or more genes to calculate a normalization factor The Normalization Factor is the mean of the selected endogenous control s which is used to normalize the Ct value of each sample Global Normalization The software first finds the common assays among all samples The median Cy of those as
20. ults C7 data exported from the following versions of Applied Biosystems software Table 1 Instrument File usta Biosystems Instrument Software Versions extension Viia 7 Real Time PCR System Viia 7 Software v1 0 amp v1 1 RQ 7900HT Fast Real Time PCR System SDS v2 2 2 2 3 amp 2 4 AQ Results 7900HT Fast Real Time PCR System SDS v2 2 2 2 3 amp 2 4 RQ Study Results files must be in the Plate Centric Table Orientation ABI PRISM 7000 Sequence Detection System SDS v1 1 amp v1 2 RQ Study Results sdm 7300 Real Time PCR System SDS v1 4 RQ Study Results sdm 7500 amp 7500 Fast Real Time PCR System SDS v1 4 RQ Study Results sdm 7500 Fast Real Time PCR System Software v2 0 RQ Study Results edm StepOne amp StepOne Plus Real Time PCR System StepOne Software v2 2 RQ Study Results edm Summary OpenArray Real time PCR Instrument OpenArray Real time PCR Software v1 0 results csv For all data from Applied Biosystems Real Time PCR instruments make sure that you have set appropriate baselines and thresholds for your experiments before exporting the data For more information on properly analyzing results please refer to the instrument user manual Note RQ data must be Results exported from an RQ study file except StepOne Software v2 0 amp OpenArray The results files are imported during the set up of a new experimental study The Sample Name Assay Name Assay Type and C values are automaticall
21. y entered into the study from the results file Helpful Shortcut Icons Create a new study Open saved study Edit a study Save a study Delete or close a study Export analysis results Help Toggle between studies sa 7oago oD Create a New Study Create a new study either by clicking on the 5 icon in tool box or File gt New Enter a Study Name in the box at the top of the window x Study Name test run Import results files see Table 1 for supported file format by clicking on the Add Files button in the New Study window then click OK Page 2 of 13 Samples will now appear in the Sample Design box and Assay information in the Assay Design box DataAssist Software v2 9 Senay Crag AOD Setings Ome r r kon kon r pac r r r E ai Gap l Maun alowable Cr wha H0 0 i i YT a dk H Omt E m inkade mue Cr vakas in caldatore ves r A Exdude culhers among replicates E r or ge bam Target ge Cad o Adaa p values uae Bergame Hochberg Fohe Dacovery Gate Crecse normakiation meted ncogenos Conta Select endogenous controle for arabes A Reference sarciejyon kaitrai SE Lal Qc Mot Bia araca Samer ot Gace Rents teamu agma er Exporting and Saving Study amp Data Files Exporting Data Files Export results data from the study by either clicking on the a icon or navigating to
22. ype box and change the assignment or upload an assay design file Click on Browse to find and select your assay design file Page 4 of 13 Analysis Settings The Analysis Settings section enables specific choices to be made regarding how the calculations are done Analysis Settings Maximum allowable Cr value 40 0 Indude max Ct values in calculations Yes kd Exdude outliers amona replicates w Adjust p values using Benjamini Hochberg False Discovery Rate 4 Choose normalization method Endogenous Control Reference sample group calibrator universal e Maximum Allowable C Value This is used as a detection threshold or C cut off value Any value above the maximum allowable is changed to the maximum allowable value This change is reflected in the Adjusted C column in the C Data tab e Include Max C Values in Calculations If Yes is selected wells with Max Cr Cy cut off are included in the analysis If you set the C cut off at C 32 everything above C 32 will be converted to 32 including wells with a Cry of 40 not detected A fold change will be calculated for each well even those which displayed no amplification C 40 If No is selected all wells with a Cy greater than the Max Cy are excluded from analysis e Exclude Outliers Among Replicates Outliers within technical replicates will be excluded from data analysis calculations see Appendix B This change is reflected in the Adjusted C column in

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