actin as internal standard in RT-PCR

JIrving jirving at aol.com
Sun Oct 8 03:48:51 EST 1995


<In semi-quantitative RT-PCR, is it valid to use beta-actin as an
internal standard when comparing the expression of a gene in DIFFERENT
TISSUES? Or is it only valid when comparing the expression of a gene in
a specific tissue at different developmental stages?

If the answer to question 1 is "no", what internal standard is best to
use?>

Don't use actin , since actin expression is cell-cycle dependent; it's
best to use GAPDH, although this also has some variability in rare cases. 
I have used GAPDH for my external standard in quantitative RT-PCR for five
years or so and it works well for me with minimal variability.  I wouldn't
recommend using it as an internal standard in the PCR reaction itself of
course, since this would give you much more variable kinetics. There have
been a number of papers in the past few years regarding this topic; let me
know if you need some references. Good luck!

John



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