Table 3 In-house developed primers and RT-PCR protocol
Primer sequences | ||
|---|---|---|
Forward 1 | 5′-ATG GGN TGG GAY TAY CC-3′ | ~ 440 bp amplicon |
Forward 2 | 5′-GGA YTA YCC NAA RTG YGA-3′ | |
Reverse | 5′-CCA TCA TCA SWN ARN ATS AT-3′ | |
Reverse transcription | |||
|---|---|---|---|
Master mix 1 constituents per reaction | Temperature profile | ||
Random hexamer | 1 µL | 65 °C On ice | 5 m 1 m |
dNTP (10 mM) | 1 µL | ||
RNA template | 8 µL | ||
Total | 10 µL | ||
Master mix 2 constituents per reaction | RT profile | ||
|---|---|---|---|
10x RT buffer | 2 µL | ||
MgCl2 (25 mM) | 4 µL | 25 °C | 10 m |
DTT (0.1 M) | 2 µL | 50 °C | 50 m |
RNaseOUT | 1 µL | 85 °C | 5 m |
SuperScript III reverse transcriptase | 1 µL | 15 °C | Hold |
Master mix 1 | 10 µL | ||
Total | 20 µL | ||
PCR | ||||
|---|---|---|---|---|
Master mix constituents per reaction | PCR profile | |||
10x PCR buffer | 5 µL | |||
MgCl2 (25 mM) | 8 µL | |||
dNTP (10 mM) | 1 µL | 95 °C | 9 m | |
Forward primer 1 (200 µM) | 1 µL | 95 °C (1 °C/s) | 1 m | |
Forward primer 2 (200 µM) | 1 µL | 48 °C (1 °C/s) | 1 m | 40× |
Reverse primer (200 µM) | 1 µL | 72 °C (1 °C/s) | 1 m | |
PCR-grade water | 11.5 µL | 72 °C | 5 m | |
AmpliTaq Gold™ | 1.5 µL | 15 °C | Hold | |
RT product | 20 µL | |||
Total | 50 µL | |||