Biology Reference
In-Depth Information
Table 3
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
Table 4
Composition of qRT-PCR reaction mix according to the TaqMan
®
technique
Component
1× Master mix (
μ
L)
H
2
O
1.75
20× TaqMan
®
MicroRNA Assay
0.75
2× TaqMan
®
Universal PCR Master Mix,
No AmoErase UNG
7.5
Table 5
Reaction setup for qRT-PCR for detection of mature miRNAs according
to the polyT adaptor technique
Component
1× Master mix (
μ
L)
2× QuantiTect SYBR Green PCR Master Mix
10
10× miScript Universal Primer
2
10× miScript Primer Assay
2
H
2
O
4
3.7 polyT Adaptor
qRT-PCR for Mature
miRNA (Qiagen)
1. Prepare a qRT-PCR reaction mix specifi c for each primer set
according to Table
5
in a 1.5 mL reaction tube. Add 10-20 %
excess volume (
see
Note 15
).
2. Cap the tube, invert several times to mix and centrifuge briefl y.
3. Transfer 18
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
.
