can i pool RNA from a group of 10 healthy controls to generate cDNA -> real time PCR (expression of the pooled sample will be the "calibrator" to perform the delta-delta Ct method)?
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Trof
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Posted 04 February 2011 - 02:02 AM
AFAIK (my experience regarding patient samples is limited) you can and you should. Using pooled normals is a common practice.
But be sure to pool each sample with the same concentration. Other thing you should do is run the pool together with each healthy control to see how big variation there is between normal samples. For example if some of them differ more then say 3 Cts from the pooled one, then any result with "diseased" sample closer than 3 Cts is not statistically important, because is within normal range of a healthy sample.
But be sure to pool each sample with the same concentration. Other thing you should do is run the pool together with each healthy control to see how big variation there is between normal samples. For example if some of them differ more then say 3 Cts from the pooled one, then any result with "diseased" sample closer than 3 Cts is not statistically important, because is within normal range of a healthy sample.
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