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.

[1]  Thomas D. Schmittgen,et al.  Real-Time Quantitative PCR , 2002 .

[2]  E. Lukhtanov,et al.  3'-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. , 2000, Nucleic acids research.

[3]  R. Dunn,et al.  Concise review: gene expression applied to toxicology. , 1999, Toxicological sciences : an official journal of the Society of Toxicology.

[4]  J. Winer,et al.  Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. , 1999, Analytical biochemistry.

[5]  D. Botstein,et al.  The transcriptional program in the response of human fibroblasts to serum. , 1999, Science.

[6]  C. V. D. Schoot,et al.  Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes , 1998, Leukemia.

[7]  C. Reading,et al.  Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards. , 1998, Cancer research.

[8]  T. B. Morrison,et al.  Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. , 1998, BioTechniques.

[9]  P. Guenthner,et al.  Quantitative, competitive PCR assay for HIV-1 using a microplate-based detection system. , 1998, BioTechniques.

[10]  É. Oswald,et al.  A reverse transcription-polymerase chain reaction method to analyze porcine cytokine gene expression. , 1997, Veterinary immunology and immunopathology.

[11]  Kirk M. Ririe,et al.  Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. , 1997, Analytical biochemistry.

[12]  R. Müller,et al.  Cell-cycle regulation of gene expression by transcriptional repression. , 1997, Trends in genetics : TIG.

[13]  E. Benveniste.  Cytokines: influence on glial cell gene expression and function. , 1992, Chemical immunology.

[14]  C. Wittwer,et al.  Continuous fluorescence monitoring of rapid cycle DNA amplification. , 1997, BioTechniques.

[15]  J. Ross,et al.  The half-life of c-myc mRNA in growing and serum-stimulated cells: influence of the coding and 3' untranslated regions and role of ribosome translocation , 1994, Molecular and cellular biology.

[16]  S. Jacob,et al.  Control of gene expression by lipophilic hormones , 1993, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[17]  H. Yokoi,et al.  Quantification of mRNA by non-radioactive RT-PCR and CCD imaging system. , 1992, Nucleic acids research.

[18]  K. Danenberg,et al.  Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction. , 1992, Cancer research.

[19]  S. Bates,et al.  Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. , 1990, Biochemistry.

[20]  A. Gazdar,et al.  Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[21]  M. Shani,et al.  The nucleotide sequence of the rat cytoplasmic β–actin gene , 1983 .

[22]  M. Shani,et al.  The nucleotide sequence of the rat cytoplasmic beta-actin gene. , 1983, Nucleic acids research.