Uniq 10 rna extraction kit

Total RNA was extracted from the placental syncytiotrophoblasts 24 h after treatment using an UNIQ-10 RNA extraction kit (Sangon Biotech, Shanghai, China). After determination of RNA concentration, mRNA was reverse transcribed to cDNA with oligo (dT) 12–18 primer using Moloney murine leukemia virus reverse transcriptase (Promega) and cDNA was utilized for subsequent measurement of HSD11B2 and SP1 mRNA levels with qRT-PCR using power SYBR green PCR master mix (Toyobo, Osaka, Japan). The annealing temperature was set at 61°C. The absolute mRNA levels in each sample were calculated according to a standard curve set up using serial dilutions of known amounts of specific PCR product templates against corresponding cycle threshold values. To control for sampling errors, qRT-PCR for the housekeeping gene ACTB was performed on each sample. The ratio of the copy numbers of target gene over ACTB in each sample was obtained to normalize the expression of the target gene. The primer sequences for amplifying human HSD11B2, SP1 and ACTB genes are given in Table 1.