Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods

Anal Biochem. 2000 Oct 15;285(2):194-204. doi: 10.1006/abio.2000.4753.


Four quantitative reverse transcription-PCR (RT-PCR) methods were compared to evaluate the time course of mRNA formation and decay. Mouse fibroblasts (NIH 3T3) transfected with the human beta-globin open reading frame/c-myc 3'-untranslated region chimeric gene under control of the c-fos promoter (fos-glo-myc) were used for serum-inducible transcription. The amount of fos-glo-myc mRNA, relative to beta-actin, was measured by quantitative, RT-PCR at various times following the addition of serum to serum-starved fibroblasts transfected with the chimeric gene. Both endpoint (band densitometry and probe hybridization) and real-time (SYBR green and TaqMan) PCR methods were used to assay the identical cDNA. The real-time methods produced a 4- to 5-log dynamic range of amplification, while the dynamic range of the endpoint assays was 1-log. The real-time and probe hybridization assays produced a comparable level of sensitivity that was considerably greater than band densitometry. The coefficient of variation from 22 replicate PCR reactions was 14.2 and 24.0% for the SYBR green and TaqMan detection, respectively, and 44.9 and 45.1% for the band densitometry and probe hybridization assays, respectively. The rank order for the values of r(2) obtained from the linear regression of the first-order mRNA decay plots was SYBR green > TaqMan > probe hybridization > band densitometry. Real-time PCR is more precise and displays a greater dynamic range than endpoint PCR. Among the real-time methods, SYBR green and TaqMan assays produced comparable dynamic range and sensitivity while SYBR green detection was more precise and produced a more linear decay plot than TaqMan detection.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells / physiology
  • Animals
  • Cells, Cultured
  • Computer Systems
  • DNA Primers / chemistry
  • Fluorescent Dyes
  • Genes, fos / genetics
  • Genes, myc / genetics
  • Globins / analysis
  • Globins / biosynthesis
  • Globins / genetics
  • Humans
  • Linear Models
  • Mice
  • RNA, Messenger / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction*
  • Sensitivity and Specificity
  • Time Factors


  • DNA Primers
  • Fluorescent Dyes
  • RNA, Messenger
  • Globins