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

Exp Mol Pathol. 2005 Aug;79(1):14-22. doi: 10.1016/j.yexmp.2005.03.004.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line, Tumor
  • DNA Primers / genetics*
  • DNA, Complementary
  • Gene Dosage*
  • Gene Expression Profiling / methods*
  • Humans
  • Nitric Oxide Synthase / genetics
  • Nitric Oxide Synthase Type III
  • RNA, Messenger / analysis*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Transcription, Genetic / genetics*
  • Transfection
  • Vascular Endothelial Growth Factor A / genetics


  • DNA Primers
  • DNA, Complementary
  • RNA, Messenger
  • Vascular Endothelial Growth Factor A
  • NOS3 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type III