Semi-quantitative reverse transcription (RT-PCR)
Reverse transcription was done with the SuperScript II One-Step RT-PCR
System (Takara) following the manufacturer’s protocols. The following
sequences were used: the Biot2 forward primer: 5’-GGATCCAAAATGAAA
TGTGCAAAGCATCC-3’ and reverse primer: 5’-CTCAGCACCAATGAAG
CCCTCTCTT-3’; and the GAPDH (used as a control) forward primer: 5’-CCC
TTCATTGACCTCAACTA-3’ and reverse primer: 5’-CCAAAGTTGTCAT
GGATGAC-3’. The following RT-PCR conditions were used: an initial
denaturation step at 50ºC for 50 min, 94ºC for 4 min, followed by 35 cycles at
94ºC for 45 sec, 58ºC for 45 sec, and 72ºC for 45 sec, and a final elongation step
at 72ºC for 10 min. The RT-PCR fragment was then cloned into pMD18-T
vectors (TaKaRa, Japan) and sequenced (Corporation Invitrogen, China).