Biology Reference
In-Depth Information
5. Store the RT reaction product at −20 °C if you do not
immediately continue with the PCR amplifi cation step.
1. Adjust RNA to a fi nal concentration of 50 ng/
μ
L and place on
3.5 cDNA Synthesis
for Precursor miRNA
( See Note 11 )
ice (
see
Notes 7
and
8
).
2. Prepare the RT master mix using the Qiagen miScript Reverse
Transcription Kit for polyT adaptor qRT-PCRs (
see
Table
3
).
3. Mix gently by pipetting up and down.
L (100 ng) of template RNA to the RT master mix
into a 0.2 mL reaction tube.
4. Keep it on ice until you are ready to load the thermal cycler.
Use the following program:
Add 2
μ
37 °C
60 min
95 °C
5 min
5. Store the RT reaction product at −20 °C if you do not imme-
diately continue with the PCR amplifi cation step.
3.6 qRT-PCR for
Mature miRNA
(TaqMan
®
)
Each cDNA sample should be analyzed in triplicate. In addition, a
target molecule for normalization should be chosen and processed
in parallel (
see
Note 12
). Non-RT (without reverse transcriptase)
and no-template controls (NTCs) are also assayed in triplicate (
see
Note 13
).
1. Dilute cDNA 1:5 (+60
L H
2
O).
2. Prepare a qRT-PCR reaction mix specifi c for each primer set
according to Table
4
in a 1.5 mL reaction tube (
see
Note 14
).
Add 10-20 % excess volume (
see
Note 15
).
3. Cap the tube, invert several times to mix and centrifuge briefl y.
4. Transfer 10
μ
L of the qRT-PCR reaction mix into one well on
a 96-well PCR plate.
5. Add 5
μ
L of diluted template cDNA (respectively, H
2
O for
NTCs) per well.
6. Seal plate and vortex carefully.
7. Briefl y centrifuge the plate at 450-500 ×
g
.
8. Load the plate into the instrument and run the qRT-PCR
using the following program:
μ
95 °C
10 min
95 °C
15 s
40x
60 °C
1 min