Expression Analysis of Telomerase-Genes hTERT and hTR by Quantitative peR on LightCycier m
• Cooling for 1 min at 40°C
Set "display mode" to fluorescence channel 2/1 (F 2/1).
Results
The fluorescence profiles and quantification of hTERT and hTR from one sample, the five standards and positive and negative controls are shown in Figs. 1
and 2.
For analysis of the data, the F2/Fl display mode was used. The quantities of the
transcripts are calculated by using the "second derivative maximum" method. We
have found a better reproducibility and a lower user dependence than using the
"fit-point" method.
We accepted only a correlation coefficient (r) of -1.00, to keep a high validity of Error and Correlation
our results.
The relative expression level is calculated by dividing the amount of hTERT or
hTR transcripts, respectively, by the amount of PBGD transcripts.
We recommend repeating the quantification at least once to validate the results.
In-House Protocol: Materials
LightCycler Instrument (Roche Diagnostics)
LightCycler Capillaries (Roche Diagnostics)
LightCycler-DNA Master SYBR Green I (Roche Diagnostics)
First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech)
peqGOLD TriFast (peqLab)
bovine pancreatic DNase I (Eurogentec)
RNAguard Ribonuclease Inhibitor, Porcine (Amersham Pharmacia Biotech)
TaqStart Antibody (Clontech, Heidelberg, Germany)
Amplification primers (Amersham Pharmacia Biotech)
Telomerase positive tissue sample or cell line, e.g., Ewing tumor cell line VH64
Procedure
Equipment
Kits
Reagents
For RNA isolation and purification of the tissue samples of interest and of the Sample Preparation
telomerase positive reference sample, see "Sample Preparation" under "Commercial Kit Protocol".
From the samples, cDNA is synthesized from approximately 2 flg of RNA using
the First-Strand cDNA Synthesis Kit from Amersham Pharmacia Biotech and
stored at -20°C.
• Cooling for 1 min at 40°C
Set "display mode" to fluorescence channel 2/1 (F 2/1).
Results
The fluorescence profiles and quantification of hTERT and hTR from one sample, the five standards and positive and negative controls are shown in Figs. 1
and 2.
For analysis of the data, the F2/Fl display mode was used. The quantities of the
transcripts are calculated by using the "second derivative maximum" method. We
have found a better reproducibility and a lower user dependence than using the
"fit-point" method.
We accepted only a correlation coefficient (r) of -1.00, to keep a high validity of Error and Correlation
our results.
The relative expression level is calculated by dividing the amount of hTERT or
hTR transcripts, respectively, by the amount of PBGD transcripts.
We recommend repeating the quantification at least once to validate the results.
In-House Protocol: Materials
LightCycler Instrument (Roche Diagnostics)
LightCycler Capillaries (Roche Diagnostics)
LightCycler-DNA Master SYBR Green I (Roche Diagnostics)
First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech)
peqGOLD TriFast (peqLab)
bovine pancreatic DNase I (Eurogentec)
RNAguard Ribonuclease Inhibitor, Porcine (Amersham Pharmacia Biotech)
TaqStart Antibody (Clontech, Heidelberg, Germany)
Amplification primers (Amersham Pharmacia Biotech)
Telomerase positive tissue sample or cell line, e.g., Ewing tumor cell line VH64
Procedure
Equipment
Kits
Reagents
For RNA isolation and purification of the tissue samples of interest and of the Sample Preparation
telomerase positive reference sample, see "Sample Preparation" under "Commercial Kit Protocol".
From the samples, cDNA is synthesized from approximately 2 flg of RNA using
the First-Strand cDNA Synthesis Kit from Amersham Pharmacia Biotech and
stored at -20°C.