qRT-PCR
Judith Sunzenauer (last update 8.7.2014)
Procedures
Reverse Transcription:
- Prepare Master mix for 1 reaction: 2µl 10x Reverse Transcription Buffer, 0.8µl 100mM dNTPs, 2µl 10x RT Random Primers, 1µl MultiScribe Reverse Transcriptase (50U/µl), 1µl RNase Inhibitor, 3.2µl nuclease-free water
- For each reaction, combine 10µl RT master mix with 10µl total RNA (200ng per reaction)
- Use the following parameter to program the thermal cycler:
| Time | Temperature |
|---|---|
| 10 min | 25 °C |
| 120 min | 37 °C |
| 5 min | 85 °C |
| ∞ | 4 °C |
- Store at -15 to -25°C
qRT-PCR
- Master mix for 1 reaction: 1µl 20X TaqMan Assay, 10µl 2X Gene Expression Master Mix, 7µl nuclease-free water
- Pipette 18µl Master mix in each well, use three replicates
- Add 2µl RT product
- 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 sec | 95 °C |
| 60 sec | 60 °C |
- Analysis and normalization according to the ddCt method
Materials
| High Capacity cDNA Reverse Transcription Kit | Applied Biosystems #4374966 |
| TaqMan Gene Expression Assays | Applied Biosystems |
| TaqMan Gene Expression Master Mix | Applied Biosystems #4369016 |
| 96-Well optical reaction plate | Applied Biosystems #4306737 |
| MicroAmp optical adhensive film | Applied Biosystems # 4311971 |
| ABI 7300 Real-Time PCR System Applied Biosystems |