Quantitative reverse transcription-polymerase chain reaction to study mRNA decay: comparison of endpoint and real-time methods.
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.
Schmittgen
T D
TD
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, Washington 99164-6534, USA. Schmittg@mail.wsu.edu
Zakrajsek
B A
BA
Mills
A G
AG
Gorn
V
V
Singer
M J
MJ
Reed
M W
MW
D016475:3T3 Cells / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D003199:Computer Systems; D017931:DNA Primers / Q000737:chemistry; D005456:Fluorescent Dyes; D016762:Genes, fos / Q000235:genetics; D016259:Genes, myc / Q000235:genetics; D005914:Globins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D016014:Linear Models; D051379:Mice; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors
D017931:DNA Primers; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D005914:Globins