Quantification of porcine follicle-stimulating hormone receptor messenger ribonucleic acid by reverse transcription-competitive polymerase chain reaction

Abstract

An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20-bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.

Keywords

follicle-stimulating hormone receptor / mRNA / reverse transcription-competitive polymerase chain reaction

References

[1] Hayward-LesterA, OefnerP J, SabatiniS, et al.. Accurate and absolute quantitative measurement of gene expression by single-tube RT-PCR and HPLC. Genome Res, 1995, 5: 494-494

[2] GauseW C, AdamoviczJ.. The use of the PCR to quantitate gene expression. PCR Methods Appl, 1994, 3: S123-S123

[3] VolkenandtM, DickerA P, BanerjeeD, et al.. Quantitation of gene copy number and mRNA using the polymerase chain reaction [Minireview]. Proc Soc Exp Biol Med, 1992, 200: 1-1

[4] GillilandG, PerrinS, BlanchardK, et al.. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA, 1990, 87: 2725-2725

[5] RappoleeD A, WangA, MarkD, et al.. Novel method for studying mRNA phenotypes in single or small numbers of cells. J Cell Biochem, 1989, 39: 1-1

[6] ChellyJ, MontarrasD, PinsetC, et al.. Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction-application to dystrophin mRNA in culture myogenic and brain cells. Eur J Biochem, 1990, 187: 691-691

[7] SantagatiS, BettiniE, AsdenteM, et al.. Theoretical considerations for the application of competitive polymerase chain reaction to the quantitation of a low abundance mRNA: estrogen receptor. Biochem Pharmacol, 1993, 46: 1797-1797

[8] GrassiG, ZentilinL, TafuroS, et al.. A rapid procedure for the quantitation of low abundance RNAs by competitive reverse transcription-polymerase chain reaction. Nucleic Acids Res, 1994, 22: 4547-4547

[9] DiviaccoS, NorioP, ZentilinL, et al.. A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates. Gene, 1992, 122: 313-313

[10] SeibelP, MellO, HannemannA, et al.. A method for quantitative analysis of deleted mitochondrial DNA by PCR in small tissue samples. Methods Mol Cell Biol, 1991, 2: 147-147

[11] Becker-AndreM, HahlbrockK.. Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY). Nucleic Acids Res, 1989, 25: 9437-9437

[12] HencoK, HeibeyM., QuantitativePCR. the determination of template copy numbers by temperature gradient gel electrophoresis (TGGE). Nucleic Acids Res, 1990, 18: 6733-6733