[Quantitation of human telomerase reverse transcriptase mRNA with real-time fluorescent RT-PCR].

AIM: To establish a real-time fluorescent RT-PCR assay to quantify human telomerase reverse transcriptase (hTERT) mRNA.

METHODS

Total cellular RNA was isolated from HepG2 cells using Trizol reagent, which was then reverse-transcribed into cDNA. By using a pair of gene-specific primers and a TaqMan MGB probe, cDNA of hTERT was quantified with real-time fluorescent PCR. Human beta-actin (hBA) mRNA was quantified at the same time as an endogenous control. Serial dilutions of cloned human beta-actin gene were used to construct a standard curve.

RESULTS

The correlation coefficient of the standard curve was 1.00. The mean coefficient of variation of the assay was 7.1%. The ratio of hTERT mRNA to hBA mRNA of HepG2 cells was (1.3+/-0.3)x10(-4).

CONCLUSION

A real-time fluorescent RT-PCR assay to quantify hTERT mRNA was established, which will facilitate the study on the biological function of telomerase.