Sybrgreen pcr kits

Analysis was performed following the Minimum Information for Publication of Quantitative Real-Time PCR Experiment guidelines as previously described [23]. Briefly, isolated RNA was reverse-transcribed using a PrimeScript™ RT Reagent Kit (RR047A; Takara, Kusatsu, Japan), and miRNA was reverse-transcribed using a miRcute Plus miRNA First-Strand cDNA Kit (KR211; Tiangen, Beijing, China). Target gene and miRNA expression levels were detected using a real-time PCR instrument (LightCycler480 II; Roche, Switzerland) and SYBRGreen PCR Kits (FP205 and FP411, respectively; Tiangen, Beijing, China). The relative expression levels of candidate genes were normalized to the reference gene (GAPDH or U6) and calculated using the comparative CT (2^−ΔΔCT) method. The primer sequences used were GAPDH: (Fw) 5′-GAC​TCA​TGA​CCA​CGT​CCA​TGC-3′ and (Rv) 5′-AGA​GGC​AGG​GAT​GAT​GTT​CTG-3′; Smad2: (Fw) 5′-GTG​TCA​CCA​TAC​CAA​GCA​CTT​GC-3′ and (Rv) 5′-GAC​TCA​AAG​CGA​CAG​ATA​ACA​CG-3′. miRNA primers were purchased from Tiangen (Beijing, China) used as commercial products. Amplification efficiencies of genes ranged from 90% to 115%, and melting curves of tested genes indicated that the amplification was specific.

qPCR was performed using a LightCycler® 480II and Tiangen SYBR Green PCR Kits. U6 was used as the endogenous control for miRNA amplification. The reaction volume was 25 μL, and was set up according to the TB GreenTM Premix Ex TaqTMII protocol. All samples and blanks were analyzed in triplicate. The reaction conditions were as follows: pre-denaturation at 95 °C for 30 s, followed by 95 °C for 5 s and 60 °C for 30 s for 40 cycles. The melting curves were generated using the following temperatures: 95 °C for 5 s and 60 °C for 1 min. Relative miRNA expression level was calculated using the 2^-△△Ct method and normalized to U6 levels.