Hello, I have a problem with my gene expression results. The results of my endogenous gene are oscillating between samples, generating false negative results. It takes about 12 hours after sample collection to begin extracting PBMC (ficoll method), and then extracting RNA (phycol-chloroform method). Do you think time may be influencing results?
I am thinking of buying the RNA stabilizer tubes - PaxGene. Could I extract the PBMC and RNA by the same method mentioned above?
Hi Caroline
Can you extract RNA from total blood and not PBMC!? In this case with 250 ul of fresh blood, you can easily go with TRIzol or Accuzol or ... to extract high quality RNA as we do.
Bit confused as to what you're doing: trizol is an RNA isolation method, ficoll is a PBMC density gradient agent, and chloroform is typically used with trizol (again to isolate RNA).
Are you saying you collect blood, isolate PBMCs 12 hours later, then use those PBMCs to prepare RNA? If so, how long between PBMC isolation and RNA extraction?
Are the times always identical between batches, or is it variable?
Also, what reference genes are you using, and how do you know they're stable?
Remember, there are a lot of steps in RNA isolation/cDNA synthesis where error can creep in, and the purpose of reference genes is to control for this. If you see variation in your reference genes, that's fine: detecting such variation is the whole point of using reference genes. It's only when the variation appears to be systematic that you need worry (i.e. all ref genes down in treated samples only, or similar).
Dear Caroline
I fully agree with John. It seemed to me that you used only one reference gene. You have to try other one to see if the variation is or isn´t systematic.
Depending on the result of this first approach, you can try to use a DNA/RNA shield (e.g. Zymo Research) to protect your samples from degradation during transport and storage.
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