Quantification of Cytokine mR As in Human Myocardial Biopsy Samples •
cDNA products directly from the total RNA using a random hexamer method
without isolating poly-A RNA.
Reverse transcription reaction for each 20-~ reaction:
Volume IJ1l1
[Final]
25 mMMgCl2
4
5mM
Deoxynucleotide
2
ImM
Random primer p(dN)6
2
0.08 Au,c U
RNase inhibitor
1
AMV reverse transcriptase
0.8
lOx Reaction Buffer
2
Milli-Q H2O
8.2
Total master mix volume per reaction
20
RT reaction
25°C
42°C
99°C
The LightCycler protocol was as follows:
• Denaturation for 30 s at 95°C
• Amplification
Parameter
Va1ue
55
Cycles
Type
Quantification
Target temperature 1°C]
Incubation time [s]
Temperature transition rate [OC/sJ
Acquisition
• Melting Curve Analysis
Segment 1
95
0
20
None
Parameter
Value
Segment 2
55- 60
5
20
None
Cycles
Type
Melting curve analysis
50 U
20 U
Ix
10 min
60 min
5 min
Segment 3
72
10
20
None
Segment 1
Segment 2
Target temperature [0C]
Incubation time [s]
Temperature transition rate [OC/s I
Acquisition
95
0
20
None
The fluorimeter gain of Channel 1 was set to 1.
60
30
20
None
Segment 4
81-83
5
20
Single
Segment 3
95
0
0.1
Cont.
Primers for quantitative PCR were designed to satisfy the following criteria: First- peR Primer Design
ly, primers were designed so that there is no complementarity between the sense
and antisense PCR primers, especially at the 3' end, thus avoiding the formation of