Expression Analysis of Telomerase-Genes hTERT and hTR by Quantitative PCR on LightCycier 1m
Table 1. Oligonucleotides
hTERT (GenBank Accession #AF018167)
Position
Length
GC (%)
Tm (OC)
5' -CGGAAGAGTGTCTGGAGCAA-3'
5' -CATGGACTACGTCGTGGGAG-3'
Product
1785
1980 R
1785-1980
20
20
196
hTR (GenBank Accession #AF047386)
5' -CCTAACTGAGAAGGGCGTAGGC-3'
5' -CTAGAATGAACGGTGGAAGGCG-3'
Product
849
961 R
849-961
22
22
lJ3
GAPDH (GenBank Accession #M33197)
5' -CACCCATGGCAAATTCCAIGGC-3'
5'-GCATTGCTGATGATCTTGAGGCT-3'
Product
213
509 R
213-509
22
23
297
55.0
60.0
59.1
54.5
54.5
47.8
60.8
61.0
66.5
65.3
67.0
65.4
glass capillary and placed in cooled adapters. The first eight capillaries are needed for one negative (H 2 0-) control and the seven dilutions of the reference sample. Set the dilutions to "standard": 1:1=100; 1:2=50; 1:4=25; 1:8=12.5; 1:16=6.25;
1:32=3.125; 1:64=1.5625. The remaining capillaries can be used for the samples of
interest. The sealed capillaries are briefly centrifuged (10 s at 700 g) and placed
into the LightCycler rotor.
The following PCR protocol is used, with conditions for GAPDH/hTERT/hTR
amplification as follows:
• Denaturation at 95°C for 2 min
• Amplification
Parameter
. Value
Cycles
Type
Target temperature rOC)
40 (GAPDH)/45 (hTERT)/40 (hTR)
Quantification
Segment 1
Segment 2
95
63 (GAPDH)I
Segment 3
60 (bTERT)/62 (hTR)
72
Incubation time [s)
Temperature transition rate (OC/s)
20
5
20
Acquisition mode
Gains
None
Single
Fl=5;F2=10;F3= 10
14
20
None