Biology Reference
In-Depth Information
7. Load the plate into the instrument and run the qRT-PCR
using the following program:
95 °C
15 min
94 °C
15 s
55 °C
30 s
40x
70 °C
30 s
It is strongly recommended to perform a melting curve
(70-85 °C, read every 0.5 °C for 3 s) analysis at the end of the
cycling program ( see Note 16 ).
1. Prepare a qRT-PCR reaction mix according to Table 6 in a
1.5 mL reaction tube. Add 10-20 % excess volume.
2. Cap the tube, invert several times to mix and centrifuge briefl y.
3. Transfer 18
3.8 qRT-PCR for
Precursor miRNA
L of the qRT-PCR reaction mix into one well on
a 96-well PCR plate.
4. Add 2
μ
μ
L of template cDNA (respectively, H 2 O for NTCs) per
well.
5. Seal plate and vortex carefully.
6. Briefl y centrifuge the plate at 450-500 × g .
7. Load the plate into the instrument and run the qRT-PCR
using the following program:
95 °C
15 min
94 °C
15 s
55 °C
30 s
40x
70 °C
30 s
Table 6
Reaction setup for qRT-PCR for detection of precursor miRNAs according
to the polyT adaptor technique
Component
1× Master mix (
μ
L)
H 2 O
6
10× miScript Precursor Assay ( see Note 17 )
2
2× QuantiTect SYBR Green PCR Master Mix
10
Search WWH ::




Custom Search