Quantitative multi-gene transcriptional profiling using real-time PCR with a master template.
We previously reported a method for quantitative multi-gene transcriptional profiling with gene-specific standard curves using real-time PCR. Here, we report an approach that increases experimental throughput by using a master template to generate a single standard curve for the estimation of mRNA copy numbers from all genes. We prepared fifty-nine different templates and measured eNOS mRNA copy numbers in Matrigel VEGF-transfectant samples. The copy numbers obtained using each of the fifty-nine templates were within 50% of the copy number obtained using the previously reported method. Analysis of primer design parameters, and subsequent tests, showed that eliminating complementarities between the first nucleotides at the 5'-ends of the forward and reverse primers reduces the influence of saturation effects and produces copy numbers similar to those generated with gene-specific templates-generally, within 20%. Measurements on a panel consisting of eNOS, iNOS, and nNOS further validated the master-template approach. The master-template approach enables rapid quantification of mRNA abundances from panels of hundreds of genes, and will be a valuable tool for screening large numbers of genes as part of a search for biomarkers, the validation of DNA-microarray data, or research into the dynamics of the gene-protein network.
Shih
Shu-Ching
SC
Pathology Department, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215, USA. sshih2@bidmc.harvard.edu
Smith
Lois E H
LE
D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics*; D018076:DNA, Complementary; D018628:Gene Dosage; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D019001:Nitric Oxide Synthase / Q000235:genetics; D052250:Nitric Oxide Synthase Type III; D012333:RNA, Messenger / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III