Biology Reference
In-Depth Information
Table 1
Composition of reverse transcription master mix according
to the TaqMan
®
technique
Component
1× Master mix
10× Reverse Transcription Buffer
1.5
μ
L
dNTPs (100 mM)
0.15
μ
L
RNase Inhibitor (20 U/
μ
L)
0.19
μ
L
MultiScribe
TM
Reverse Transcriptase (50 U/
μ
L)
1
μ
L
Reverse Transcription primer (
see
Note 10
)
3
μ
L
Nuclease-free H
2
O
ad 13
μ
L
Table 2
Composition of reverse transcription master mix according
to the polyT adaptor technique
Component
1× Master mix
5× miScript RT Buffer
4
μ
L
miScript Reverse Transcriptase Mix
1
μ
L
Nuclease-free H
2
O
ad 18
μ
L
3. Mix gently by pipetting up and down.
Add 2
μ
L (10 ng) of template RNA to 13
μ
L (TaqMan
®
) or
L (Qiagen) RT mastermix, respectively, into a 0.2 mL
reaction tube.
4. Keep it on ice until you are ready to load the thermal cycler.
Stem-loop qRT-PCR (TaqMan
®
): Before loading the thermal
cycler, incubate samples 5 min on ice. Use then the following
program:
18
μ
16 °C
30 min
42 °C
30 min
85 °C
5 min
PolyT adaptor qRT-PCR (Qiagen): Use the following
program (preincubation is not required):
37 °C
60 min
95 °C
5 min
