Superscript riii first strand kit

Total RNA was extracted from E17 chick phalanges and MC3T3-E1 cells using a Trizol kit (Invitrogen, Waltham, MA, USA). RNA (1 μg) was reverse transcribed, using 1 μL Oligo dT, 1 μL StarScript II RT mix, primer, and 10 μL 2× reaction mix, according to the manufacturer’s instructions (Genstar, Beijing, China). First-strand cDNA (0.4 μL) was synthesized to a final volume of 20 μL using a SuperScript RIII first-strand kit (Invitrogen, Waltham, MA, USA). Following reverse transcription, PCR amplification of the cDNA was performed using mouse/chick specific primers. The primers’ sequences are provided in Table S1. The PCR reactions were performed in a Bio-Rad S1000TM Thermal cycler (Bio-Rad, Hercules, CA, USA) as described previously [54 (link)]. The expression of the genes was normalized to GAPDH, and the expression levels were compared by ΔΔCt. The qPCR results shown are representative of three independent experiments.

Total RNA was extracted from human placentas and HTR8 cell lines using a TRIzol kit (Invitrogen, USA). First-strand cDNA was synthesized to a final volume of 20 µl using a SuperScript RIII first-strand kit (Invitrogen, USA). Following reverse transcription, PCR amplification of the cDNA was performed using the corresponding specific primers (note: the sequences of the primers are provided in Supplementary file 1: Table S2). Primers were obtained from Sangon Biotech (Shanghai, China). q-PCR was performed using 2× RealStar Fast SYBR qPCR Mix (A304-10, GenStar, China) in a Bio-Rad S1000TM Thermal cycler (Bio-Rad, USA). The reaction conditions were as follows: 95 °C for 2 min, 40 cycles at 95 °C for 15 s and 60 °C for 30 s, melt curve 65 to 95 °C, increment 0.5 for 5 s. The target gene mRNA expression levels were normalized using 18 S as a reference. The results were analyzed using the 2^−ΔΔCt method.