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

doi:10.1006/abio.2000.4753

Analytical Biochemistry

Volume 285, Issue 2, 15 October 2000, Pages 194-204

https://doi.org/10.1006/abio.2000.4753 Get rights and content

Abstract

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.

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^☆: Supported by Grants CA74375-01 (NIH) and IRG-119Q (American Cancer Society) to T.D.S.

²: To whom reprint requests should be addressed. Fax: (509) 335-5902. E-mail: [email protected].

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

Abstract

Trends Genet.

Vet. Immunol. Immunopathol.

Anal. Biochem.

Anal. Biochem.

Concise review: gene expression applied to toxicology

Toxicol. Sci.

Control of gene expression by lipophilic hormones

FASEB J.

Cytokines: Influence on glial cell gene expression and function

Chem. Immunol.

Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression

Biochemistry

Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction

Proc. Natl. Acad. Sci. USA

Quantification of mRNA by non-radioactive RT-PCR and CCD imaging system

Nucleic Acids Res.

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