Multiplex Relative RT-PCR Method for Verification of Differential Gene Expression
Year: 1999
Type: article
Abstract: Differential display, suppression subtractive hybridization and other techniques for identification of differentially expressed genes produce fragments of cDNA from mRNAs whose differences in abundance must be verified. This report describes a relative multiplex RT-PCR assay that facilitates the analysis of large numbers of samples for differences in mRNA abundance without the use of radioactivity or blotting. The species of interest is co-amplified with 18S rRNA over a range of cycles followed by electrophoresis through ethidium bromide-agarose gels. Intensities of the bands of interest, normalized for 18S band intensities, are plotted as a function of cycle number. Regression equations fitted to the curves are used to calculate the number of cycles necessary for each sample's normalized signal to reach a threshold intensity. Differences between samples in the number of cycles required to reach that threshold reflect differences in the original abundances of those species. A comparison with results previously obtained using northern blots showed that relative differences as small as 20% and as large as an order of magnitude are accurately detected. The simplicity of the assay allows its routine application in both research and teaching laboratories.
Source: BioTechniques
DOI: 10.2144/99275rr04
Authors: Weston E. Spencer, Merrill J. Christensen
Institution: Brigham Young University
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $1,415
OpenAlex ID: W2160547845