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
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