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Biotechniques. 1998 Apr;24(4):652-8.

Reproducibility in the quantification of mRNA levels by RT-PCR-ELISA and RT competitive-PCR-ELISA.

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University of Leicester, Department of Pathology, Leicester Royal Infirmary, England, UK.


The use of reverse transcription (RT) PCR for relative quantitation of gene transcripts relies on the reproducibility of the individual RT, PCR and product measurement steps. Semi-competitive RT-PCR (RT-cPCR) uses an internal competitor template in the PCR step to improve quantitation. We have surveyed the reproducibility of RT, PCR, RT-cPCR and measurement, amplifying the glyceraldehyde-3-phosphate dehydrogenase "housekeeping" gene from isolated renal glomeruli. We used an enzyme-linked immunosorbent assay (ELISA) to quantify PCR products. We also report our PCR-based method for constructing a competitor DNA identifiable independently of the native product. Our results show that the entire RT-PCR and ELISA process had a standard deviation (SD) of less than 10% (n = 10). This compared to an SD of less than 13% (n = 10) in PCR and ELISA. The SD for ELISA alone was less than 11% (n = 10). RT-cPCR quantitation gave an SD of approximately 15% (n = 10). These results support the use of standard RT-PCR for the relative quantitation of mRNA. RT-cPCR is also suited to relative quantitation, but it is also independent of the amplification saturation curve and permits the identification of differences in cellularity between samples.

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