m Applications in Oncology
Kits LightCycler Telo TAGGGG hTERT Quantification Kit (Roche Diagnostics)
LightCycler Telo TAGGGG hTR Quantification Kit (Roche Diagnostics)
Reagents
peqGOLD TriFast (peqLab, Erlangen, Germany)
Sample Preparation
Oligonucleotide
Design
LightCycier PCR
bovine pancreatic DNase I (Eurogentec, Seraing, Belgium)
RNAguard Ribonuclease Inhibitor, Porcine (Amersham Pharmacia Biotech, Freiburg, Germany)
Procedure
RNA is isolated from fresh or frozen tissue samples (solid or cell culture) using
peqGOLD TriFast in accordance with the manufacturer's protocol. The RNA is
purified by DNase I digestion and resuspended in DEPC-treated water to obtain
a final RNA concentration between 100 and 2,000 ng/~, which is measured spectrophotometrically.
For analysis, the RNA is diluted to 50 ng/I.ll for hTR quantification and
50-100 ng/I.ll for hTERT quantification, as recommended in the manufacturer's
protocol.
Telomerase (hTERT)-encoding mRNA and telomerase-associated RNA (hTR) is
reverse transcribed and a fragment of the generated cDNA is amplified with
specific primers. The amplicon is detected by fluorescence using a specific pair
of hybridization probes. The hybridization probes consist of two different short
oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the amplification cycle. One probe is
labeled at the 5'-end with LightCycler Red 640, and to avoid extensions, modified at the 3' -end by phosphorylation. The other probe is labeled at the 3' -end
with fluorescein.
The sequences of the primers and probes are not published.
The primers for hTERT and porphobilinogen desaminase (PBGD) are specific
for mRNA because their sequences span exon splice junctions. Co amplification of
contaminating genomic DNA is therefore prevented. However, as hTR is an
intron-free gene, the primers also amplify genomic DNA. Because of this, controls
without reverse transcriptase (RT) are strongly recommended, to make sure that
there is no contaminating DNA amplified (see hTR Master Mix).
We have found that RNA purification by DNase I digestion eliminates the contaminating DNA sufficiently, so the reverse transcriptase negative samples did not
produce a signal. Therefore, we do not currently perform these controls on a regular basis.
Each Quantification Kit uses PBGD as a housekeeping gene, which is simultaneously quantified with the gene of interest in a "one-step RT-PCR".
For quantification, five standards of in-vitro transcribed hTERT or hTR RNA
are provided with concentrations within the linear range of the reaction from 10 2
to 10 6 copies, or from 10 3 to 10 7 copies, respectively, per 2 ~. The exact number of
Kits LightCycler Telo TAGGGG hTERT Quantification Kit (Roche Diagnostics)
LightCycler Telo TAGGGG hTR Quantification Kit (Roche Diagnostics)
Reagents
peqGOLD TriFast (peqLab, Erlangen, Germany)
Sample Preparation
Oligonucleotide
Design
LightCycier PCR
bovine pancreatic DNase I (Eurogentec, Seraing, Belgium)
RNAguard Ribonuclease Inhibitor, Porcine (Amersham Pharmacia Biotech, Freiburg, Germany)
Procedure
RNA is isolated from fresh or frozen tissue samples (solid or cell culture) using
peqGOLD TriFast in accordance with the manufacturer's protocol. The RNA is
purified by DNase I digestion and resuspended in DEPC-treated water to obtain
a final RNA concentration between 100 and 2,000 ng/~, which is measured spectrophotometrically.
For analysis, the RNA is diluted to 50 ng/I.ll for hTR quantification and
50-100 ng/I.ll for hTERT quantification, as recommended in the manufacturer's
protocol.
Telomerase (hTERT)-encoding mRNA and telomerase-associated RNA (hTR) is
reverse transcribed and a fragment of the generated cDNA is amplified with
specific primers. The amplicon is detected by fluorescence using a specific pair
of hybridization probes. The hybridization probes consist of two different short
oligonucleotides that hybridize to an internal sequence of the amplified fragment during the annealing phase of the amplification cycle. One probe is
labeled at the 5'-end with LightCycler Red 640, and to avoid extensions, modified at the 3' -end by phosphorylation. The other probe is labeled at the 3' -end
with fluorescein.
The sequences of the primers and probes are not published.
The primers for hTERT and porphobilinogen desaminase (PBGD) are specific
for mRNA because their sequences span exon splice junctions. Co amplification of
contaminating genomic DNA is therefore prevented. However, as hTR is an
intron-free gene, the primers also amplify genomic DNA. Because of this, controls
without reverse transcriptase (RT) are strongly recommended, to make sure that
there is no contaminating DNA amplified (see hTR Master Mix).
We have found that RNA purification by DNase I digestion eliminates the contaminating DNA sufficiently, so the reverse transcriptase negative samples did not
produce a signal. Therefore, we do not currently perform these controls on a regular basis.
Each Quantification Kit uses PBGD as a housekeeping gene, which is simultaneously quantified with the gene of interest in a "one-step RT-PCR".
For quantification, five standards of in-vitro transcribed hTERT or hTR RNA
are provided with concentrations within the linear range of the reaction from 10 2
to 10 6 copies, or from 10 3 to 10 7 copies, respectively, per 2 ~. The exact number of