Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Anal Biochem. 1994 Dec;223(2):251-8.

Monitoring mRNA expression by polymerase chain reaction: the "primer-dropping" method.

Author information

  • 1Department of Medical Biochemistry, University of Calgary, Alberta, Canada.

Abstract

We have developed a method to monitor mRNA expression that is based upon the reverse transcriptase-polymerase chain reaction (RT-PCR) and includes multiple sets of primer pairs in coamplification reactions. To observe relative changes in mRNA steady-state levels, each target in a multiplex reaction was amplified to within a predetermined range by using PCR cycle numbers specific for each target. Optimal PCR cycle numbers for target templates were determined by preliminary titration experiments performed using the "primer-dropping" method. By employing this method, the overall amplification reaction was limited, permitting the PCR products to remain within the exponential range of the amplification curve and yet be detectable on ethidium bromide-stained gels. We demonstrated the utility of this method by monitoring the expression kinetics of cyclins A, B1, D1, and E, and of the immediate-early genes c-fos, c-myc, and beta-actin. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included in the multiplex reactions as an endogenous internal standard to control for variations in product abundances due to differences in individual RT and PCR reaction efficiencies. Changes in gene expression of less than twofold to greater than 75-fold were readily distinguished.

PMID:
7887471
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Write to the Help Desk