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  • Title: Quantification of estrogen receptor messenger RNA by quantitative polymerase chain reaction using internal standard fragment.
    Author: Kagami I, Mizunuma H, Miyamoto S, Ibuki Y, Uchida T.
    Journal: Biochem Biophys Res Commun; 1996 Nov 12; 228(2):358-64. PubMed ID: 8920919.
    Abstract:
    A simple and reliable polymerase chain reaction-based method for quantifying human and rat estrogen receptor (ER)-mRNA is described. This method involves co-amplification of cDNA transcribed from sample RNA with an internal standard to reduce tube to tube amplification variations. Human and rat internal standards were synthesized by insertion of a DNA fragment near the midportion of human and rat target ER cDNA molecules. After co-amplification, both products of target ER cDNA and internal standard were separated on agarose gel by electrophoresis and transferred onto a membrane filter. The radioactivity hybridized with 32P-labeled ER cDNA was counted. The ER mRNA content was calculated by linear regression analysis after obtaining a logarithm of ratios between the sample and the internal standard radioactivity. The coefficient of variation of this assay was 18.8%. An absolute amount of ER mRNA in less than 10(4) cultured cells could be measured.
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