I am currently trying to analyze my RT-qPCR data and I am hearing differing methods on how to properly perform the analysis.
I have 3 technical replicates for each of my biological samples and I have heard that if the Ct standard deviation between my three technical replicates is 0.5 or greater, I can remove the "outlier" (the technical replicate that has a Ct value that deviates the furthest from the other two) and re-analyze the Ct average and Ct standard deviation from the remaining two technical replicates. Is this a common method for performing qPCR analysis? Or is there a different, commonly accepted Ct standard deviation threshold that is used to remove outliers? Or is there a separate statistical test?
My standard deviation between technical replicates is typically below 0.09, however in some cases there is one technical replicate that is an "obvious outlier", but I'm questioning whether there is a common rule that will allow me to remove this outlier.
Thanks for your help!
You can use the outlier labeling rule by Tukey: upper limit = Q3+(Q3-Q1)*g and lower limit = Q1-(Q3-Q1)*g. Q stands for quartile. For more information please review the following YouTube page:
https://www.youtube.com/watch?v=WSflSmcNRFI
Run your data on the outlier detector.
http://graphpad.com/quickcalcs/Grubbs1.cfm
Since there are several technical issues independent to the biological difference that might be present associated with CT differences (PCR reaction efficiency, pippeting errors, etc) I don't think is a good practice to perform analysis on just two technical replicates. There are researchers that actually suggest a technical quadruplicate.
Here are a couple of resources for you to review
there was a very nice thread asociated with this topic before and I think it articulates the points I am trying to make even better. https://www.researchgate.net/post/Triplicate_for_samples_in_qPCR_analysis1 Also: https://www.thermofisher.com/content/dam/LifeTech/Documents/PDFs/PG1503-PJ9169-CO019879-Re-brand-Real-Time-PCR-Understanding-Ct-Value-Americas-FHR.pdf
Also is a good idea to read the manual and have it to troubleshoot in the future.
http://find.thermofisher.com/qpcr/real-pcr-handbook/merch
I hope it helps.
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