Quantitative Reverse Transcription–Polymerase Chain Reaction to Study mRNA Decay: Comparison of Endpoint and Real-Time Methods

• Published in: Analytical biochemistry Vol. 285; no. 2; pp. 194 - 204
• Main Authors: Schmittgen, Thomas D., Zakrajsek, Brian A., Mills, Alan G., Gorn, Vladimir, Singer, Michael J., Reed, Michael W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-10-2000
• Subjects: 3T3 Cells - physiology; Animals; Cells, Cultured; Computer Systems; DNA Primers - chemistry; endpoint PCR; Fluorescent Dyes; gene expression; Genes, fos - genetics; Genes, myc - genetics; Globins - analysis; Globins - biosynthesis; Globins - genetics; Humans; Linear Models; Mice; mRNA stability; real-time PCR; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Sensitivity and Specificity; SYBR green; TaqMan; Time Factors; endpoint PCR; real-time PCR; SYBR green; TaqMan; gene expression; mRNA stability
• ISSN: 0003-2697; 1096-0309
• 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 β-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 β-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 r2 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.