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

Ying Guo, Jun Liu, Caifang Xue, Jing Wu, Tianbao Song

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Abstract

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.

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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.

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Available abstract

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.

Key concepts: Telomerase reverse transcriptase, Reverse transcriptase, Molecular biology, Trizol, TaqMan, Serial dilution, Complementary DNA, Real-time polymerase chain reaction

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