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Exp Mol Pathol. 2005 Aug;79(1):14-22.

Quantitative multi-gene transcriptional profiling using real-time PCR with a master template.

Author information

1
Pathology Department, Beth Israel Deaconess Medical Center, Harvard Medical School, 99 Brookline Avenue, Boston, MA 02215, USA. sshih2@bidmc.harvard.edu

Abstract

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.

PMID:
15894312
DOI:
10.1016/j.yexmp.2005.03.004
[Indexed for MEDLINE]

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