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
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