Revertaid first strand dna synthesis kit

Total RNA from HepG2.2.15, HepAD38, HepG2 or HuH7 cells was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's protocol, and treated with DNase I (Thermo Fisher Scientific, Waltham, MA). The isolated RNA was reverse‐transcribed using RevertAid First Strand DNA Synthesis Kit (Thermo Fisher Scientific). The primers used for qRT‐PCR are shown in Table S2.
The reaction was carried out in a MicroAmp Optical 96‐well plate using SYBR Green PCR Master Mix (Roche, Mannheim, Germany), with 1 μL cDNA in each well. PCRs were monitored in real‐time using the Roche Lightcyler 480II Real‐time PCR System (Roche). The procedures of the PCR were 95°C for 5 min; 95°C for 20 sec, 60°C for 20 sec, and 72°C for 20 sec, 40 cycles; and 72°C for 7 min. The mRNA of the ACTB (β‐actin) gene was used as the internal control.

Quantification of HBV RNA in THLE-2 cells was performed as previously described [24 (link)]. HBV RNA was isolated using the EasyPure Viral RNA Kit (TransGen Biotech, Beijing, China) and reverse transcribed using RevertAid First Strand DNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). The levels of HBV RNA were detected by quantitative real-time PCR with a SYBR Green or TaqMan probe method using LightCycler 480 II Real-time PCR Detection System (Roche, Mannheim, Germany).
The level of HBV DNA was quantified by artus HBV PCR Kits CE (QIAGEN) according to the manufacturer’s instructions.

The RNA extraction was performed utilizing TRIzol reagent (Invitrogen, Carlsbad, CA, USA) in compliance with manufacturer’s protocols. The RevertAid™ First Strand DNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for reverse transcription. qRT-PCR was conducted with QuantiFast SYBR-Green PCR kit (Roche, Indianapolis, IN, USA) on ABI 7300 Real-time PCR System (Applied Biosystems, Foster City, CA, USA) with cDNA working as the template. The gene relative expressions were determined utilizing 2^-ΔΔCT analysis, and the primers were synthesized by BGI (Shenzhen, China) with GAPDH and U6 working as the endogenous controls.

Total RNA was extracted from cell lines or tissues using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was reverse transcribed into cDNA using a RevertAid™ First Strand DNA Synthesis kit (Thermo Fisher Scientific, Inc.). QuantiFast SYBR-Green PCR kit (Roche Diagnostics) was used to perform qPCR on an ABI 7300 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling protocol was as follows: Initial denaturation at 95°C for 5 min, followed by 45 repeats of a three-step cycling program consisting of 10 sec at 95°C (denaturation), 10 sec at 60°C (primer annealing) and 10 sec at 72°C (elongation), and a final extension step for 10 min at 72°C. The sequences of the primers were as follows: GATA6-AS1, forward 5′-ACCACAACCACTACCTTATGGCGT-3′, reverse 5′-TGCCATCTGGACTGCTGGACAATA-3′; miR-19a-5p, forward 5′-GTTTGCTGGGAAGGCAAAG-3′, reverse 5′-TGTTTTGCTGGGAAGGCAAA-3′; U6, forward 5′-CGCTTCGGCAGCACATATAC-3′, reverse 5′-TTCACGAATTTGCGTGTCAT-3′; TET2, forward 5′-GGACTGAGCTGCTGAATTCAACT-3′, reverse 5′-CCTCAACATGGTTGGTTCTATCC-3′; and GAPDH, forward 5′-TGTCCGTCGTGGATCTGA-3′ and reverse 5′-TTGCTGTTGAAGTCGCAGGAG-3′. The relative expression levels of GATA6-AS1, miR-19a-5p and TET2 were normalized to endogenous controls GAPDH or U6 and were expressed as 2^−ΔΔCq (20 (link)).

The viral RNA was extracted from 200 µl of serum using the TRIzol method following the manufacturer’s instructions in the Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan. Two hundred microliters serum sample was admixed with TRIzol. After mixing and short incubation, the same volume of chloroform was added and centrifuged at 13,000 rpm for 15 min. Upper layer was separated and mixed with Isopropanol in equal volume. After centrifugation, the supernatant was discarded and the pellet was collected. To the pellet, 300 µl chilled ethanol was added and incubated on ice. Pellet was dried and 100 µl sterile distilled water was added in pellet for further use. The quantity of RNA was determined by a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized using Revert Aid First Strand DNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.

Total RNA was extracted from cells and tissues using the TRIzol reagent (Invitrogen,
Carlsbad, CA, USA). The subcellular fractions of NSCLC cells were separated using the
PARIS Kit (Ambion, Austin, TX, USA). RevertAid™ First Strand DNA Synthesis Kit (Thermo
Fisher Scientific, Waltham, MA, USA) was used for reverse transcription, and quantitative
real-time polymerase chain reaction (qRT-PCR) was executed using an SYBR Green PCR Kit
(Toyobo, Osaka, Japan) on the 7500 Fast Dx Real-Time PCR Instrument (Applied Biosystems,
Foster City, CA, USA). β-actin was considered as a control for
normalization. MicroRNA detection was conducted using a miDETECT A Track Kit (RiboBio,
Guangzhou, China). The small nuclear RNA U6 expression was employed as a
control for normalization. The primers for this research were designed using Primer
Premier 5 software. The sequences are presented in Table S1 (See Supplementary Online
Information at www.celljournal.org).