Assessing the expression of two genes simultaneously in surgical specimens using polymerase chain reaction.
We developed a novel polymerase chain reaction (PCR)-based method to analyze simultaneously the relative expression of two genes in a single PCR reaction. The method, relational PCR (R-PCR), utilizes special PCR primers that enable a PCR reaction to be converted from a standard uniplex reaction to a multiplex reaction in which all products are dependent on the same reaction components for amplification. We show that the quantitative ability of R-PCR is unaffected by sample nucleic acid input concentration over a range of 25-fold (30 to 750 ng of total RNA) and demonstrate excellent interexperimental reproducibility. We used R-PCR to analyze estrogen receptor gene expression in a series of invasive breast carcinomas, and our results show an excellent correlation between estrogen receptor mRNA expression and protein product accumulation determined by standard immunocytochemistry on paraffin sections.
Shepard
S B
SB
Pathology Department, Faulkner Hospital, Boston, Massachusetts 02130, USA.
Cooper
A G
AG
D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007633:Keratins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012334:RNA, Neoplasm / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013048:Specimen Handling
D017931:DNA Primers; D018076:DNA, Complementary; D012334:RNA, Neoplasm; D011960:Receptors, Estrogen; D007633:Keratins