Fastking rt kit with gdnase kr116

Total RNA was isolated from the tissues preserved in liquid nitrogen using the RNA extraction kit RN38 (Aidlab Biotech, China). RNA integrity was identifed with a 1.2% agarose gel, and RNA quantity and quality were determined by NanoDrop 1000C Spectro-photometer (Thermo Scientific, USA). The cDNA was obtained by reverse transcription of total RNA using the FastKing RT Kit (With gDNase) KR116 (TIANGEN, China) and used for qRT-PCR by SYBR® rapid quantitative PCR Kit (KAPA KK4601, USA). Gene-specific primers are listed in Table S2 with products lengths between 100 bp and 300 bp. Tcactin was used as a reference gene. Relative abundance of TcMYBs were calculated according to the comparative Cq method described in previous study (Li & Lu, 2014 (link); Ma et al., 2012 (link)). The expression levels of miR159, miR828 and miR858 were analyzed using the method as described previously (Shi & Chiang, 2005 (link)). One-way ANOVA was calculated using IBM SPSS 19 software. P < 0.01 was considered statistically significant and was represented by asterisks. The heat maps of differential expression of TcMYB genes were performed using HemI 1.0 with gradient bar (Clustering Method is Average linkage and Similarity Metric is Pearson distance (default)) (Deng et al., 2014 (link)).

For expression analyses, 30-day old Sujiang piglets from different families were randomly selected and genotyped for GHR-RIP10. Seven female piglets for SINE^+/+genotype and six female piglets for SINE^+/− were selected. Liver, kidney, leg muscle, longissimus dorsi, and back fat were collected after slaughter and quick-frozen by liquid nitrogen and then stored at −80 °C. The mRNA was extracted using standard Trizol methods (Invitrogen, Carlsbad, CA, USA). The first strand of cDNA was prepared according to the manufacturer’s protocol by using FastKing RT Kit (With gDNase, KR116) (TIANGEN, Beijing, China). Then, the mRNA expression of GHR was evaluated by quantitative real-time PCR (qPCR) using the qTower3G PCR System (Analytik Jena AG, Thuringia, Germany) in a total volume of 20 μL according to the manufacturer’s instructions (TaKaRa SYBR Premix Ex Taq^TM, Dalian, China). GAPDH was used as an endogenous control. The relative expression level of the gene was calculated by formula 2^−ΔΔCt based on the qPCR results. The specificity of the qPCR products was checked on 1.5% ethidium bromide-stained agarose gels and further confirmed using melting curve analyses.