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An alternative quantitative polymerase chain reaction method.

Analytical Biochemistry

Transcription, Genetic, Reproducibility of Results, Reference Values, Rats, Inbred BN, Rats, analysis, RNA, Messenger, methods, Polymerase Chain Reaction, Molecular Sequence Data, Models, Statistical, Male, metabolism, Kidney, Glyceraldehyde-3-Phosphate Dehydrogenases, genetics, Erythropoietin, Blotting, Northern, Base Sequence, Animals, Anemia

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      Abstract

      The exponential nature of the polymerase chain reaction (PCR) makes quantitation of amplified products possible only if the efficiency of the enzymatic steps is estimated or cast off. To obtain a relative quantitative measurement of the reverse transcription (RT)-PCR products, a series of seven progressive dilutions achieved by mixing RNA solutions of two different samples to be compared was prepared in a constant final volume. An aliquot of each dilution mix was submitted to a standard RT-PCR. This range of concentrations allowed the elimination of the tube-to-tube efficiency variations. Indeed, after gel densitometric analysis of the amplified products, the alignment of the seven measurements along a regression line demonstrated that the PCR efficiencies in all tubes was equal allowing a direct comparison between the two samples. To illustrate this method, the renal erythropoietin (EPO) expression level was compared in anemic and control rats. Reverse transcription was performed using specific primers for EPO and GAPDH genes. The EPO mRNA expression was also checked by Northern blotting. Quantitative PCR indicated that anemic rats produced 19 times more EPO mRNA than did control rats. The results from Northern blotting matched with those of PCR. This simple new method does not provide absolute amounts of nucleic acid but relative ones, and it works with any set of primers. It could be used alternatively to methods such as competitive RT-PCR.

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      Journal
      10.1006/abio.1996.0161
      8660499

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