Rapid competitive PCR determination of relative gene expression in limiting tissue samples.
Reverse transcriptase (RT)-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of procedures, quantification of RT-PCR products remains difficult, particularly when processing a large number of samples. Therefore, we developed a novel alternative PCR technique that we term "rapid competitive PCR" (RC-PCR), designed to study the relative expression of specific genes in a large number of small tissue biopsies. RC-PCR is characterized by measuring relative gene expression at the mRNA level of two or more samples with a nonradioactive assay based on competitive PCR amplification between identical sequences of internal standard and target cDNA. Only a single reaction tube per sample is used in this technique, and it was validated by comparing RC-PCR of protein kinase C zeta and alpha expression in rat colonic mucosa samples with competitive RT-PCR analysis (requiring 6-8 reaction tubes per sample). We conclude that RC-PCR is a simple, rapid, highly sensitive technique that is capable of detecting less than twofold differences in mRNA expression.
Jiang
Y H
YH
Faculty of Nutrition, Molecular and Cell Biology Group, Texas A&M University, College Station, 77843-2471, USA.
Davidson
L A
LA
Lupton
J R
JR
Chapkin
R S
RS
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D001706:Biopsy; D003106:Colon / Q000201:enzymology; D015870:Gene Expression; D007413:Intestinal Mucosa / Q000201:enzymology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011493:Protein Kinase C / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D015203:Reproducibility of Results
D012333:RNA, Messenger; D011493:Protein Kinase C; D012194:RNA-Directed DNA Polymerase