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mRNA quantification and clinical evaluation of telomerase reverse transcriptase subunit (hTERT) in intracranial tumours of patients in the island of Crete.

Yannopoulos A, Dimitriadis E, Scorilas A, Trangas T, Markakis E, Talieri M - Br. J. Cancer (2005)

Bottom Line: Telomerase is a reverse transcriptase that maintains telomeres by adding telomeric TTAGGG repeats to the ends of human chromosomes.Patients having negative expression of hTERT mRNA had statistically longer PFS (P=0.031) and OS (P=0.047).Cox univariate regression analysis revealed that hTERT mRNA-positive patients had a high and statistically significant risk of relapse (hazard ratio (HR) of 2.24 and P=0.038).

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, University Hospital of Heraclio, Heraclio, Crete, Greece.

ABSTRACT
Telomerase is a reverse transcriptase that maintains telomeres by adding telomeric TTAGGG repeats to the ends of human chromosomes. The aim of this study was to evaluate quantitatively the mRNA expression of telomerase catalytic subunit (hTERT) in different types of intracranial tumours in relation to their histologic pattern and grade and correlate it with progression-free (PFS) and overall survival (OS) of patients. Human telomerase reverse transcriptase mRNA levels were estimated by the use of real time RT-PCR in 68 samples of intracranial tumours. It revealed statistical correlation between hTERT mRNA expression levels and the grade of the tumours (P<0.001). Patients having negative expression of hTERT mRNA had statistically longer PFS (P=0.031) and OS (P=0.047). Cox univariate regression analysis revealed that hTERT mRNA-positive patients had a high and statistically significant risk of relapse (hazard ratio (HR) of 2.24 and P=0.038). In the Cox multivariate regression model, the levels of hTERT mRNA were adjusted for tumour grade and patients age, and since there was statistically significant relationship between the levels of hTERT mRNA and the grade of the tumours (P=0.003 or P=0.006, respectively), hTERT mRNA levels could not be considered as an independent prognostic factor for PFS or OS.

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Example of real-time RT–PCR quantification of hTERT mRNA in tumour samples. Top, hTERT standard curve; bottom, hTERT analysis (htertst1, 2, 3, 4, 5 are human TERT RNA Standards with concentration of 7.3 × 105, 7.0 × 104,8, 5 × 103, 8.4 × 102, 8.1 × 101 copies per 2 μl, respectively; Positcontrol=positive control RNA; negat=negative control; samples 1–11 correspond to intracranial tumour samples).
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fig1: Example of real-time RT–PCR quantification of hTERT mRNA in tumour samples. Top, hTERT standard curve; bottom, hTERT analysis (htertst1, 2, 3, 4, 5 are human TERT RNA Standards with concentration of 7.3 × 105, 7.0 × 104,8, 5 × 103, 8.4 × 102, 8.1 × 101 copies per 2 μl, respectively; Positcontrol=positive control RNA; negat=negative control; samples 1–11 correspond to intracranial tumour samples).

Mentions: In short description, telomerase (hTERT)-encoding mRNA was reversed transcribed and a 198 bp fragment (corresponding to the full-length hTERT transcript) of the generated cDNA was amplified with specific primers in a one-step RT–PCR reaction. The amplicon was detected by fluorescence using a specific pair of hybridisation probes. The hybridisation probes consist of two different short oligonucleotides that hybridise to an internal sequence of the amplified fragment during the annealing phase of the amplification cycle. One probe is labelled at the 5′-end with LightCycler-Red 640, and to avoid extension, modified at the 3′-end by phosphorylation. The other probe is labelled at the 3′-end with fluorescein. The emitted fluorescence of LightCycler-Red 640 was then measured at the instrument LightCycler. In a separate one-step RT–PCR, mRNA, encoding for porphobilinogen deaminase (PBGD), was processed for use as a house-keeping gene. The reaction product served both as a control for RT–PCR performance and as a reference for relative quantification. During initial optimisation of PCR conditions, amplified products were analysed using agarose gels to ensure correct product size. Once the PCR product was determined with Light Cycler melting-curve analysis, it was used to control for the specificity of amplifications. The number of transcripts in samples was calculated with the LightCycler software, using the calibration data obtained from serial dilutions of purified PCR products containing known numbers of cDNA molecules of each gene (Figure 1). The results of real-time PCR were given as the ratio between hTERT and PBGD transcripts, expressed as a percentage and converted to arbitrary units dividing by 1000. All experiments were performed in duplicate, with good consistency of results (the mean coefficient of variation was 9.5%). All samples were analysed in a blind-trial fashion.


mRNA quantification and clinical evaluation of telomerase reverse transcriptase subunit (hTERT) in intracranial tumours of patients in the island of Crete.

Yannopoulos A, Dimitriadis E, Scorilas A, Trangas T, Markakis E, Talieri M - Br. J. Cancer (2005)

Example of real-time RT–PCR quantification of hTERT mRNA in tumour samples. Top, hTERT standard curve; bottom, hTERT analysis (htertst1, 2, 3, 4, 5 are human TERT RNA Standards with concentration of 7.3 × 105, 7.0 × 104,8, 5 × 103, 8.4 × 102, 8.1 × 101 copies per 2 μl, respectively; Positcontrol=positive control RNA; negat=negative control; samples 1–11 correspond to intracranial tumour samples).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361474&req=5

fig1: Example of real-time RT–PCR quantification of hTERT mRNA in tumour samples. Top, hTERT standard curve; bottom, hTERT analysis (htertst1, 2, 3, 4, 5 are human TERT RNA Standards with concentration of 7.3 × 105, 7.0 × 104,8, 5 × 103, 8.4 × 102, 8.1 × 101 copies per 2 μl, respectively; Positcontrol=positive control RNA; negat=negative control; samples 1–11 correspond to intracranial tumour samples).
Mentions: In short description, telomerase (hTERT)-encoding mRNA was reversed transcribed and a 198 bp fragment (corresponding to the full-length hTERT transcript) of the generated cDNA was amplified with specific primers in a one-step RT–PCR reaction. The amplicon was detected by fluorescence using a specific pair of hybridisation probes. The hybridisation probes consist of two different short oligonucleotides that hybridise to an internal sequence of the amplified fragment during the annealing phase of the amplification cycle. One probe is labelled at the 5′-end with LightCycler-Red 640, and to avoid extension, modified at the 3′-end by phosphorylation. The other probe is labelled at the 3′-end with fluorescein. The emitted fluorescence of LightCycler-Red 640 was then measured at the instrument LightCycler. In a separate one-step RT–PCR, mRNA, encoding for porphobilinogen deaminase (PBGD), was processed for use as a house-keeping gene. The reaction product served both as a control for RT–PCR performance and as a reference for relative quantification. During initial optimisation of PCR conditions, amplified products were analysed using agarose gels to ensure correct product size. Once the PCR product was determined with Light Cycler melting-curve analysis, it was used to control for the specificity of amplifications. The number of transcripts in samples was calculated with the LightCycler software, using the calibration data obtained from serial dilutions of purified PCR products containing known numbers of cDNA molecules of each gene (Figure 1). The results of real-time PCR were given as the ratio between hTERT and PBGD transcripts, expressed as a percentage and converted to arbitrary units dividing by 1000. All experiments were performed in duplicate, with good consistency of results (the mean coefficient of variation was 9.5%). All samples were analysed in a blind-trial fashion.

Bottom Line: Telomerase is a reverse transcriptase that maintains telomeres by adding telomeric TTAGGG repeats to the ends of human chromosomes.Patients having negative expression of hTERT mRNA had statistically longer PFS (P=0.031) and OS (P=0.047).Cox univariate regression analysis revealed that hTERT mRNA-positive patients had a high and statistically significant risk of relapse (hazard ratio (HR) of 2.24 and P=0.038).

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, University Hospital of Heraclio, Heraclio, Crete, Greece.

ABSTRACT
Telomerase is a reverse transcriptase that maintains telomeres by adding telomeric TTAGGG repeats to the ends of human chromosomes. The aim of this study was to evaluate quantitatively the mRNA expression of telomerase catalytic subunit (hTERT) in different types of intracranial tumours in relation to their histologic pattern and grade and correlate it with progression-free (PFS) and overall survival (OS) of patients. Human telomerase reverse transcriptase mRNA levels were estimated by the use of real time RT-PCR in 68 samples of intracranial tumours. It revealed statistical correlation between hTERT mRNA expression levels and the grade of the tumours (P<0.001). Patients having negative expression of hTERT mRNA had statistically longer PFS (P=0.031) and OS (P=0.047). Cox univariate regression analysis revealed that hTERT mRNA-positive patients had a high and statistically significant risk of relapse (hazard ratio (HR) of 2.24 and P=0.038). In the Cox multivariate regression model, the levels of hTERT mRNA were adjusted for tumour grade and patients age, and since there was statistically significant relationship between the levels of hTERT mRNA and the grade of the tumours (P=0.003 or P=0.006, respectively), hTERT mRNA levels could not be considered as an independent prognostic factor for PFS or OS.

Show MeSH
Related in: MedlinePlus