Hairpin it mirnas qpcr quantitation kit

We isolated total RNA using Trizol reagent (Invitrogen, USA) following the manufacturer’s instructions. In order to detect the expression of miR-371 in invasive (up) and non-invasive (bottom) Hep3B or HepG2 separated by the Transwell method, we used the Hairpin-it^™ miRNAs qPCR Quantitation kit (GenePharma) for reverse transcription and quantitative PCR with U6 small nuclear RNA (snRNA) as an internal control for expression normalization. The thermal cycling conditions set for miR-371 and U6 snRNA were: 95°C for 3 min, 38 cycles of 94°C for 12 sec, and 63°C for 1 min. We examined the expressions of SOX2, OCLN, LEF1, AGO1, COX15, PTEN, and ELK4 in Hep3B or HepG2 transfected with miR-371 or NC, and the expression of PTEN in Hep3B cells transfected with PTEN overexpression vector or empty vector by real-time PCR using the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and SYBR Premix Ex Taq II (2 ×) (Tli RNaseH Plus, TaKaRa, Otsu, Japan). GAPDH mRNA levels were used for normalization. The relative expression of miR-371, snRNAs, or mRNAs was normalized using the comparative threshold cycle (2^−ΔCT) method. The primer sequences used in this study were listed in the Supplementary Table S2.

Total RNA was extracted from cells (48 h post-transfection) or tissue using TRIzol^® reagent (Thermo Fisher Scientific, Inc.), and subsequently transcribed into cDNA using PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan) at 42˚C for 15 min. qPCR was performed using SYBR^® Premix Ex Taq™ kit (Takara Bio, Inc.) on an ABI 7300 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with customized primer sets for TGFBR2, CK18, E-cadherin, FN and α-SMA. The relative expression of mRNA in each sample was normalized to the level of GAPDH using the 2^-ΔΔCq method (27 (link)). The expression of miR-155 was examined using the Hairpin-it™ miRNAs qPCR Quantitation kit (Shanghai GenePharma Co., Ltd.) according to manufacturer's protocol, and U6 was used as an internal control for normalization. Primers were synthesized by GenScript and the sequences are listed in Table II.

Total RNA was extracted using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instruction, and reverse transcribed using the PrimeScript RT Reagent Kit (Takara, Dalian, People’s Republic of China). Expression of miR-194 was analyzed using Hairpin-it miRNAs qPCR Quantitation Kit (GenePharma, Shanghai, People’s Republic of China). Expression of U6 snRNA (GenePharma) and GAPDH (Takara) was used as endogenous control. qRT-PCR was performed with the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA). The primers of candidate genes are listed in Table 1. All experiments were performed in at least triplicate, and the relative expression levels were calculated using the 2^−ΔΔCt method.