Quantitation of Gene Expression by RT-PCR and HPLC Analysis of PCR Products
Part of the
Methods in Molecular Biology
book series (MIMB, volume 193)
The quantitative measurement of specific mRNA species is of major importance for studies on gene expression. Northern blotting is a relatively insensitive method requiring microgram amounts of RNA. It is time consuming and semi-quantitative at best. Because of the limitations of Northern blotting, various strategies have been developed for quantitation of cDNA by polymerase chain reaction (PCR)-based methods (1, 2, 3, 4). Competitive PCR, in which a synthetic segment is co-amplified together with the target segment, is one of these approaches. Besides adding more steps to the experimental protocol, competitive PCR is limited because the competitor and target sequences are not necessarily amplified with the same efficiency, which impairs reliable quantitation (5). Recently, real-time PCR, in which the generated PCR-products are quantified fluorimetrically after each cycle, has become widely used (6). However, determination of fragment size for positive fragment identification is not—at least not directly—possible with this method.
KeywordsHPLC Phenol DMSO Manganese Acetonitrile
Horikoshi T., Danenberg K., and Volkenandt M. (1993) Quantitative measurement of relative gene expression in human tumors. Methods Mol. Biol.
, 177–188.Google Scholar
Becker-Andre M., and Hahlbrock K. (1989) Absolute quantitation using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res.
, 9437–9446.PubMedCrossRefGoogle Scholar
Gilliland G., Perrinn S., and Blanchard K., Bunn F. (1990) Analysis of cytokine mRNA and DNA: Detection and quantitation by competitive polymerase chain reaction. Proc. Nat’l Acad. Sci. USA
, 2725–2729.CrossRefGoogle Scholar
Wang A.M., Doyle M.V., and Mark D.F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Nat’l Acad. Sci. USA.
, 9717–9721.CrossRefGoogle Scholar
Schweitzer B., Horikoshi T., et al. (1992) Quantitation of gene copy number and mRNA using the polymerase chain reaction. Proc. Soc. Exp. Biol. Med.
, 1–6.PubMedGoogle Scholar
Heid C.A., Stevens J., Livak K.J., and Williams P.M. (1996) Real time quantitative PCR. Genome Res.
, 986–994.PubMedCrossRefGoogle Scholar
Young K., Resnick R.M., and Myers T. W. (1993) Detection of hepatitis Cvirus RNA by a combined reverse transcription-polymerase chain reaction assay. J. Clin. Microbiol.
, 882–886.PubMedGoogle Scholar
Mulder J., McKinney M., Christopherson C., Sninsky J., Greenfield L., and Kwok S. (1994) Rapid and simple PCR assay for quantitation of human immunodeficiency virus type I RNA in plasma: Application to acute retroviral infection. J. Clin. Microbiol.
, 292–300.PubMedGoogle Scholar
Myers T.W., and Gelfand D.H. (1991) Reverse transcription and DNA amplification by a Thermus thermophilus DNA polymerase. Biochemistry
, 7661–7665.PubMedCrossRefGoogle Scholar
Huber C.G., Oefner P.J., Preuss E., and Bonn G.K. (1993) High resolution liquid chromatography of DNA fragments on non-porous poly(styrenedivinylbenzene) particles. Nucleic Acids Res.
, 1061–1066.PubMedCrossRefGoogle Scholar
Oefner P.J., Bonn G.K., Huber C.G., and Nathakarnkitkool S. (1992) Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA. J. Chromatogr.
, 331–340.PubMedCrossRefGoogle Scholar
Chirgwin J.M., Przybyla A.E., MacDonald R.J., and Rutter W.J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry
, 5294–5299.PubMedCrossRefGoogle Scholar
Oefner P.J., Huber C.G., Puchhammer-Stockl E. Umlauft F., Gruenewald K., Bonn G.K., et al. (1994) High performance liquid chromatography for routine analysis of hepatitis C virus cDNA/ PCR products. BioTechniques
, 898–908.PubMedGoogle Scholar
Huber C.G., Oefner P.J., and Bonn G.K. (1995) Rapid and accurate sizing of DNA fragments by ion pair chromatography on alkylated non-porous poly(styrene-divinylbenzene) particles. Anal. Chem.
, 578–585.CrossRefGoogle Scholar
Huber C.G., Oefner P.J., and Bonn G.K. (1993) High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene divinylbenzene co-polymers. Anal. Biochem.
, 351–358PubMedCrossRefGoogle Scholar
Huber C.G., Oefner P.J., and Bonn G.K. (1993) Rapid analysis of biopolymers on modified non porous polystyrene divinylbenzene particles. Chromatographía
, 653–658.CrossRefGoogle Scholar
Lehmann J.M., Hoffmann B., and Pfahl M. (1991) Genomic organisation of the retinoic acid receptor gamma gene. Nucleic Acids Res.
, 573–578.PubMedCrossRefGoogle Scholar
Huber C.G. (1998) Micropellicular stationary phases for high-performance liquid chromatography of double-stranded DNA. J. Chromatogr.
, 3–30.CrossRefGoogle Scholar