qRT-PCR of miRNA
2-step qRT-PCR
Procedures
Reverse Transcription:
- Dilute total RNA to the concentration of 2ng/µl
- Prepare Master mix for 1 reaction: 0,15µl 100mM dNTPs, 1µl MultiScribe Reverse Transcriptase (50U/µl), 1,5µl 10x Reverse Transcription Buffer, 0,19µl RNase Inhibitor (20U/µl), 4,16µl Nuclease-free water
- For each 15µl RT reaction, combine the RT master mix with total RNA in the ratio of: 7µl RT master mix : 5µl total RNA (1 to 10ng per reaction)
- Add 3µl of 5X RT primer from each assay set into the corresponding RT reaction tube
- Use the following parameter to program the thermal cycler:
| Time | Temperature |
|---|---|
| 30 min | 16 °C |
| 30 min | 42 °C |
| 5 min | 85 °C |
| ∞ | 4 °C |
- Store at -15 to -25°C
qRT-PCR:
- Master mix for 1 reaction: 1µl 20X TaqMan Small RNA Assay, 1,33µl RT Product, 10µl 2X TaqMan Universal PCR Master Mix II, 7,67µl Nuclease-free water
- Pipette 20µl Master mix in each well, use three replicates
- Seal the plate with the appropriate cover and centrifuge
- Use the following parameter to program the thermal cycler:
| Step | Time | Temperature |
|---|---|---|
| Hold | 10 min | 95 °C |
| Cycle | 15 s 60 s |
95 °C 60 °C |
- Analysis and normalization according to the ddCt method
Materials
| TaqMan MicroRNA Reverse Transcription Kit | Applied Biosystems #4366597 |
| TaqMan MicroRNA Assays | Applied Biosystems |
| TaqMan Universal PCR Master Mix II | Applied Biosystems # 4440047 |
| 96-Well optical reaction plate | Applied Biosystems #4306737 |
| Optical Caps (8 Caps/Strip) | Applied Biosystems #4323032 |
| MicroAmp optical adhensive film | Applied Biosystems # 4311971 |
| ABI 7300 Real-Time PCR System Applied Biosystems |