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Quantitative PCR to evaluate small amounts of BCL2 mRNA in human peripheral T cells: implication of equimolar target
Hiroshi Sato1, Kazue Ozawa, Keiko Yonemura
1Division of Bioscience, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan. satoh@belle.shiga-med.ac.jp
Background:
It is difficult to reliably and reproducibly quantitate small amounts of mRNA by conventional PCR. The method we describe here enabled us to more accurately quantitate a small amount of BCL2 mRNA in human peripheral T cells.
Methods:
The sample was amplified in four tubes containing three-fold serial dilutions of competitor so that when the PCR products in the four tubes were totaled, the number of target and competitor components were approximately equal. An unknown concentration of target molecules should be assessed only within a narrow range, requiring that several reactions be performed for each sample.
Results And Conclusion:
With this method, conventional PCR machines can be used to perform quantitative PCR on very small amounts of BCL2, and would be especially useful for samples that contain substance(s) that affect PCR amplification efficiency.
