Expression Analysis of Telomerase-Genes hTERT
and hTR by Quantitative PCR on LightCycier
BERND FRODERMANN, CHRISTOPHER POREMBA *
Introduction
Telomerase activity (TA) has been shown to be a strong indicator of cellular
malignancy in virtually all malignant tumors [1-5]. Telomerase describes a
ribonucleoprotein polymerase which uses an internal RNA component as a template to synthesize telomeric DNA directly onto the ends of chromosomes [6].
These repetitive sequences are considered to be important in the protection and
replication of chromosomes. Cells without TA display progressive shortening of
telomeric repeats with each cell division because lagging-strand DNA synthesis
at the very end of linear chromosomes cannot be completed. Induction of TA in
tumor cells could give rise to clonal immortality by compensating for the loss of
telomeric DNA and thus maintaining telomere length. Recently, the catalytic
subunit hTERT (or hTRT, hEST2, TP2) of human telomerase was cloned, and
detection of hTERT expression by RT-PCR revealed a strong correlation with TA
by the TRAP assay in the majority of tumors so far examined [7]. Other components of the telomerase holoenzyme complex, such as human telomerase RNA
(hTR) and telomerase protein 1 (TPl, TLPl, hTEPl), seem to be expressed in
both normal and tumor tissues, and expression levels of these genes revealed no
or only limited correlation with TA [8,9].
In this chapter, we describe two methods for quantitative real-time RT-PCR
to analyze hTERT and hTR RNA expression on the LightCycler: the first
method is based on the commercially available hTERT and hTR quantification
kits from Roche Diagnostics, whereas the second method is based on our own
protocol [10] that we developed and published before commercial kits became
available.
Commercial Kit Protocol: Materials
LightCycler instrument (Roche Diagnostics, Mannheim, Germany)
LightCycler Capillaries (Roche Diagnostics)
* Christopher Poremba (~) (e-mail: poremba@uni-muenster.de)
Gerhard-Domagk-Institute of Pathology, Westfiilische Wilhems-University, Domagkstrasse 17,
48149 Munster, Germany
Equipment
and hTR by Quantitative PCR on LightCycier
BERND FRODERMANN, CHRISTOPHER POREMBA *
Introduction
Telomerase activity (TA) has been shown to be a strong indicator of cellular
malignancy in virtually all malignant tumors [1-5]. Telomerase describes a
ribonucleoprotein polymerase which uses an internal RNA component as a template to synthesize telomeric DNA directly onto the ends of chromosomes [6].
These repetitive sequences are considered to be important in the protection and
replication of chromosomes. Cells without TA display progressive shortening of
telomeric repeats with each cell division because lagging-strand DNA synthesis
at the very end of linear chromosomes cannot be completed. Induction of TA in
tumor cells could give rise to clonal immortality by compensating for the loss of
telomeric DNA and thus maintaining telomere length. Recently, the catalytic
subunit hTERT (or hTRT, hEST2, TP2) of human telomerase was cloned, and
detection of hTERT expression by RT-PCR revealed a strong correlation with TA
by the TRAP assay in the majority of tumors so far examined [7]. Other components of the telomerase holoenzyme complex, such as human telomerase RNA
(hTR) and telomerase protein 1 (TPl, TLPl, hTEPl), seem to be expressed in
both normal and tumor tissues, and expression levels of these genes revealed no
or only limited correlation with TA [8,9].
In this chapter, we describe two methods for quantitative real-time RT-PCR
to analyze hTERT and hTR RNA expression on the LightCycler: the first
method is based on the commercially available hTERT and hTR quantification
kits from Roche Diagnostics, whereas the second method is based on our own
protocol [10] that we developed and published before commercial kits became
available.
Commercial Kit Protocol: Materials
LightCycler instrument (Roche Diagnostics, Mannheim, Germany)
LightCycler Capillaries (Roche Diagnostics)
* Christopher Poremba (~) (e-mail: poremba@uni-muenster.de)
Gerhard-Domagk-Institute of Pathology, Westfiilische Wilhems-University, Domagkstrasse 17,
48149 Munster, Germany
Equipment