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      Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

      Journal of Molecular Endocrinology

      Society for Endocrinology

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          Abstract

          The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).

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          Author and article information

          Journal
          Journal of Molecular Endocrinology
          Society for Endocrinology
          0952-5041
          1479-6813
          October 1 2000
          October 1 2000
          : 169-193
          Article
          10.1677/jme.0.0250169
          11013345
          © 2000

          Free to read

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