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


CONCLUSIONS
A real-time fluorescent RT-PCR assay to quantify hTERT mRNA was established, which will facilitate the study on the biological function of telomerase.
Guo
Ying
Y
Department of Obstetrics and Gynecology, Second Hospital of Xi'an Jiaotong University, Xi'an 710001, China.
Liu
Jun
J
Xue
Cai-fang
CF
Wu
Jing
J
Song
Tian-bao
TB
D000199:Actins / Q000032:analysis / Q000235:genetics; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics*; D005260:Female; D018197:Hepatoblastoma / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D006801:Humans; D008113:Liver Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019098:Telomerase / Q000032:analysis / Q000235:genetics*
D000199:Actins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D019098:Telomerase