Analysis of human telomerase reverse transcriptase mRNA (hTERT) expression in myxoid liposarcomas using LightCycler real-time quantitative reverse transcriptase-polymerase chain reaction

Abstract

We describe a convenient, nonradioactive reverse transcription--polymerase chain reaktion (RT-PCR) method for the rapid and accurate quantitative detection of the human telomerase catalytic subunit human telomerase reverse transcriptase (hTERT) mRNA. The LightCycler TeloTAGGG hTERT Quantification Kit (Roche Molecular Biochemicals) was designed to be used for the highly sensitive and quantitative detection of hTERT mRNA relative to the house-keeping gene porphobilinogen deaminase (PBGD). As a tumor progression model, we investigated 26 myxoid liposarcomas (11 pure myxoid grade I, 15 myxoid/round cell grade II/III) for the hTERT expression level and compared the results of the new method with former measurements performed in silver-stained polyacrylamide gels. Both methods revealed similar results, with real-time RT-PCR being the more accurate quantification technique, which also saves time and material. Elevated hTERT expression (cut-off ratio x 100 at 1.3) was an indicator of round cell components and hence for tumor progression in myxoid liposarcoma. The new method is capable of differentiating between pure myxoid and myxoid/round cell liposarcomas for hTERT-expression more accurately.

MDM2 gene amplification: a new independent factor of adverse prognosis in non-small cell lung cancer (NSCLC)

“…On the other hand, real-time PCR LightCycler technology is considered comparable to previously used techniques. Furthermore, in addition to the advantage of saving tissue material and time, this method allows more reproducible results to be achieved at a higher sensitivity [33]. Other possible reasons for higher rates of MDM2 gene in this series include ethnic and environmental factors, as well as different smoking exposure (in our population 112 of 116 patients were current or previous smokers).…”

Quantitative evaluation of telomerase subunits in urine as biomarkers for noninvasive detection of bladder cancer

“…Accurate and sensitive detection of telomerase subunits, i.e., only those hTERT splice variants that translate to a putative functionally active telomerase enzyme, was carried out using a standardized 1-step RT-PCR protocol as described. 29 Analysis of PCR kinetics and calculation of quantitative data were performed using the LightCycler Software (Roche Applied Science). Sufficient reliability of the PCR assays is indicated by intraand interassay coefficients of variation (CV) of <10% for repetitive determinations of hTERT and hTR concentrations of >10 3 copies (Biochemica Newsletter No.…”

Telomerase Activity and Telomerase Subunit Gene Expression Levels Are Not Related in Prostate Cancer: A Real-Time Quantification and In Situ Hybridization Study

“…The difference in the frequency of hTERT positivity between the current series and the one of Latil et al (2000) could be explained by different real-time RT-PCR techniques or sampling bias. Good validation of the kit we used is documented by others (Schneider-Stock et al, 2001;Yi et al, 2001). With regard to sampling, the study of Latil et al (2000) also included tissue samples from patients who received androgen ablation therapy before radical prostatectomy.…”

“…Considering other solid tumors, investigations on gastrointestinal (Nakamura et al, 1999) and cervical tumors (Wisman et al, 2001) yielded similar results demonstrating that hTERT was not applicable as a prognostic factor. This, however, contrasts with reports demonstrating high expression levels of hTERT to be correlated with aggressiveness of breast cancer (Bieche et al, 2000;Poremba et al, 2002), liposarcoma (Schneider-Stock et al, 2001), non-small cell lung cancer (Wang et al, 2002), and colorectal cancer (Gertler et al, 2002) and with recurrence risk of Wilms tumors (Dome et al, 1999).…”