Relative Quantification run files need to be imported into a Relative ...

Analysis with SDS2.21. AnalysisAnalysis Settings2. In the Ct Analysis Section of the Analysis settings Window:- from the "Detector:" drop down menu select "All Detectors"- Choose "Automatic Ct" (this will also default you to"Automatic Baseline"). We recommend that you use theautomated Ct and baseline features for optimum data quality,but you can also choose to set each of these manually.3. In the Gene Expression Section:- choose the proper sample1 from the "Calibrator Sample:" dropdown menu.- Choose the proper detector2 from the "Endogenous ControlDetector:" drop down menu (Only detectors defined asEndogenous controls in the task section of the original runfile will be available).- If you wish your exported data to contain the Ct values aswell as the analysis, check the "Export Individual ΔCts" box.- Left click Apply then left click OK.4. AnalysisAnalyze5. FileExport:- From the "Export:" drop down menu, select "Results Table".- In the "From:" section check "All wells" to export data fromall the wells containing data of "Selected Wells" to exportdata from highlighted wells only.- In the "Format:" section select SDS2.2.- Give the file a proper name and make sure the "Files of type:"drop down menu is set to "Tab-delimited Text(*.txt)".- Left click the Export button.This text file should be imported into MS Excel before further use.The data can be copied and pasted or the text file itself can be openedwith (and saved as) an Excel file. From this exported file you shouldhave the individual Ct values (if you choose to export them, see step 3above) as well as the analysis. The RQ column displays the calculatedlevel of gene expression for the replicate group associated with thetest sample. If it is greater than 1, this is a positive fold changeof that value. If the RQ is less than 1, you must divide negative 1 bythat value to obtain the true negative fold change value. This can bedone by entering the following formula into a cell in the Excel file(replace A1 with whichever cell contains the RQ value.=IF(A1

Analysis with "QPCR analysis template.xls"If you choose to do your calculations without the aid of SDS2.2 youwill still have to use SDS2.2 to export the individual Ct values asfollows:1. AnalysisAnalysis Settings2. In the Ct Analysis Section of the Analysis settings Window:- from the "Detector:" drop down menu select "All Detectors"- Choose "Automatic Ct" (this will also default you to"Automatic Baseline"). We recommend that you use theautomated Ct and baseline features for optimum data quality,but you can also choose to set each of these manually.3. In the Gene Expression Section, check the "Export IndividualΔCts" box.4. Left click Apply then left click OK.5. AnalysisAnalyze6. FileExport:- From the "Export:" drop down menu, select "Results Table".- In the "From:" section check "All wells" to export data fromall the wells containing data of "Selected Wells" to exportdata from highlighted wells only.- In the "Format:" section select SDS2.2.- Give the file a proper name and make sure the "Files of type:"drop down menu is set to "Tab-delimited Text(*.txt)".- Left click the Export button.We recommend importing the data into MS Excel before further use. Thedata can be copied and pasted or the text file itself can be openedwith (and saved as) a MS Excel file. From this exported file youshould have the individual Ct values, ignore the analysis table. Thesevalues can be copied and pasted as appropriate into the "QPCR analysistemplate.xls". This spreadsheet will automatically calculate the RQ aswell as the fold change values. The derivation and explanation of theformulas used in this sheet are explained in ABI User Bulletin#2.1 The calibrator sample serves as the basis for the comparative resultsas gene expression levels in samples are calculated relative to geneexpression levels in the calibrator sample.2 Endogenous control targets are typically constitutive RNA or DNAsequences that are present at a statistically consistent level in allexperimental samples. By using the Endogenous control as an activereference, the data from the amplification of the targets can benormalized for differences in the amount of total nucleic acid added toeach reaction