Format

Send to

Choose Destination
See comment in PubMed Commons below
J Biol Chem. 1992 Oct 25;267(30):21830-8.

Standardization of mRNA titration using a polymerase chain reaction method involving co-amplification with a multispecific internal control.

Author information

1
Sanofi Recherche, Montpellier, France.

Abstract

We describe a simplified and reliable polymerase chain reaction-based method for assaying RNAs of low abundancy. The technique involves the co-amplification of cellular RNA-derived cDNA with a multispecific cDNA of synthetic origin added as an internal standard, using primer pairs common to both templates. We show that the co-amplified templates accumulate in a parallel manner throughout both the exponential and nonexponential phases of amplification, even when the starting amounts of the templates differ by up to 2 orders of magnitude. This finding means that preliminary experiments designed to determine either the late exponential region or the amplification efficiency for each pair of primers are unnecessary. This has enabled us to develop a greatly simplified quantitation protocol. We illustrate our approach by quantifying the effect of the immunosuppressor cyclosporin A on the accumulation of interleukin-4, interferon-gamma, and interleukin-2 receptor mRNAs in phytohemagglutinin-stimulated human peripheral blood mononuclear cells.

PMID:
1400493
[Indexed for MEDLINE]
Free full text
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire
    Loading ...
    Support Center