Establishment of quantitative reverse transcription--polymerase chain reaction assays for human telomerase-associated genes.
Telomerase is an enzyme that synthesizes and adds repetitive telomeric sequences of (TTAGGG)n to the ends of chromosomes. Recently, several telomerase-associated genes have been cloned, making it possible to study the expression of these genes. Quantitative comparisons of the expression of these genes and of telomerase activity might help clarify the regulation of telomerase activity. Therefore, we established the validity of a quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for the human telomerase catalytic subunit (hTERT) mRNA and telomerase associated protein (TEP1) mRNA using the TaqMan fluorogenic detection system. Using this assay, we quantitated hTERT mRNA and TEP1 mRNA expression in two human pancreatic cancer cell lines, AsPC-1 and PANC-1. Our results indicated that the levels of hTERT mRNA and TEP1 mRNA expression in AsPC-1 were 1.50 and 2.31 times higher than in PANC-1 cells. This TaqMan RT-PCR assay appears to be useful in determining the quantities of hTERT and TEP1 mRNAs in clinical specimens. Taken together, our results indicate that it is possible to measure the expression of the major telomerase genes subunits. Furthermore it is possible to apply this technique to determine the amount of other types of mRNA.
Yajima
T
T
Department of Laboratory Diagnosis, Sapporo Medical University School of Medicine, Japan.
Yagihashi
A
A
Kameshima
H
H
Furuya
D
D
Kobayashi
D
D
Hirata
K
K
Watanabe
N
N
D002352:Carrier Proteins / Q000235:genetics; D017931:DNA Primers; D015342:DNA Probes; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006801:Humans; D012333:RNA, Messenger / Q000032:analysis*; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D019098:Telomerase / Q000032:analysis* / Q000235:genetics; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D017931:DNA Primers; D015342:DNA Probes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C104342:TEP1 protein, human; D019098:Telomerase