qPCRforum.com : Discuss GenEx and qPCR
Forum for discussion about GenEx and qPCR (quantitative real-time pcr)
Here we discuss the use and processing of inter-plate calibrators
Moderator: MultiD Support
3 posts • Page 1 of 1
I am screening 8 samples with many different genes on multiple plates. On each plate I ran two duplicate reactions with one control sample on one control gene as my interplate calibrator (two wells on each plate). How do I enter the calibrator CTs into the data matrix so that I can run interplate calibrator normalization using the data editor tool on Genex?
Thank you very much!!
To compensate for variations between runs do as follows:
1) Collect all data in the same file, with genes as columns and samples as rows. If you initially have the runs in separate files, you can use the “Merge” function in the “Grid” curtain window in the Editor
2) Enter the Interplate Calibrators (IPCs) as any other samples. Since they were run in duplicates, there will be two rows for each interplate calibrator.
3) Since you analyzed each interplate calibrator for a single gene only, enter the same CT value for all genes.
4) Select Normalize with Interplate Calibrators in the “Pre-processing” menu and pair regular samples and interplate calibrators from the same run by selecting the samples in the left window and the appropriate interplate calibrator (both replicates) in the right window. Press the select button,. The repeat the process for the next plate. Once all samples and interplate calibrators have been selected, the “Apply” button is activated. Press it for normalization.
For more information, see the “Pre-process data for relative quantification” viewlet on: http://www.multid.se/demonstration.html
Thank you very much for the reply. In my case, since I have exact the same samples but different genes on each plate, therefor, I modified (transposed) your instructions. Instead of normalizing to samples I did on genes. Basically, for each plate I created two columns for the duplicate IPC CT values (entered the same CT value for all the samples), then transposed the data before normalization so that the genes can be normalized to corresponding IPCs. It seems logically sound, and appears working.
3 posts • Page 1 of 1
Who is online
Users browsing this forum: No registered users and 2 guests
Home of the GenEx analysis software