Quantitative Measurement of the mRNA £Xpression of the Turnor-Associated Antigen PRAME If1iI
Table 1. Oligonucleotides
BLAST Search Accession # NM_006115. NM _003194 and XM_037235
Position
Length
GC(%)
Tm (0C)
Primers-PRAME (NM_OO6115)
GTCCTGAGGCCAGCCTAAGT
207
20
60
GGAGAGGAGGAGTCIACGCA
I039R
20
61
Primers-TBP (NM_003194)
CACGAACCACGGCACTGATT
861
20
55
TTTTCTTGCTGCCAGTCTGGAC
949R
22
50
Primers-~-actin (XM_037235)
GCATCGTGATGGACTCCG
522
18
61
GCTGGAAGGTGGACAGCGA
1133R
19
63
Table 2. LightCycler RT-PCR: parameters of amplification
PRAME
~-actin
TBP
Initial Denaturation (min)
10
10
Denaturation (5)
5
15
Annealing (5)
5
10
Elongation (5)
40
25
Cycles
40
40
Table 3. LightCycler RT-PCR: temperatures for amplification
10
15
10
20
40
PRAME
/l-actin
TBP
loitial Denaturation (min)
94
95
95
Denaturation (5)
95
95
95
Annealing (5)
64
68
60
Elongation (5)
72
72
72
RT -peR master mix:
Volume [j!I)
[Final]
LighlCycler-DNA FastSt.art kit
Primers (10 f1M each)
MgCl2 (25 mM)
2
I (each)
0.8
Probes (see figures for concentration)
0.5
Distilled water (peR grade)
14.7
IX
0.5 JlM
2.25 mM
65.0
61.7
64.8
64.6
61.9
66.0