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