title: Multiplex relative RT-PCR method for verification of differential gene expression.
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.
authors.affiliation: Brigham Young University, Provo, UT, USA.
authors.forename: W E
authors.initials: WE
authors.lastname: Spencer
authors.forename: M J
authors.initials: MJ
authors.lastname: Christensen
mesh_terms: D000818:Animals; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D004996:Ethidium; D015870:Gene Expression; D005979:Glutathione Peroxidase / Q000235:genetics; D007453:Iodide Peroxidase / Q000235:genetics; D008297:Male; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D051140:Selenoproteins
chemical_list: D017931:DNA Primers; D011506:Proteins; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D051140:Selenoproteins; D007453:Iodide Peroxidase; D005979:Glutathione Peroxidase; D004996:Ethidium