Talent qpcr premix sybr green kit

Columbia blood agar base, brain heart infusion (BHI), Mueller–Hinton (MH) agar, and H. pylori selective supplement (Dent) SR0147E were obtained from Oxoid, United Kingdom. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS), Hank’s balanced salt solution (HBSS), MEM nonessential amino acids, TRIzol reagent, and Live/Dead BacLight Bacterial Viability kits (Molecular Probes) were purchased from Thermo Fisher Scientific, United States. Sheep blood was procured from Pingrui Biotechnology, China. CLR was purchased from Dalian Meilun Biotechnology, China. MTZ and saponin were acquired from Sigma, Germany. Fastking gDNA Dispelling RT SuperMix Kit, Talent qPCR PreMix (SYBR Green) Kit, TIANamp Bacteria DNA kit, and 2 × Taq PCR Mix were purchased from TIANGEN, China. Anti-H. pylori antibody ab20459 was obtained from Abcam, United Kingdom. Alexa Fluor 488 and goat anti-rabbit were bought from Southern Biotech, United States.

Nine genes were screened out for RNA-seq validation using quantitative real-time PCR (qRT-PCR) with Talent qPCR Premix (SYBR Green) kit (TIANGEN Biotech Co., Ltd., Beijing) according to the manufacturer’s instructions. cDNA was produced from 2 μg total RNA using FastKing gDNA Dispelling RT Supermix (TIANGEN Biotech Co., Ltd., Beijing). Primers (supplementary Table 1) for qRT-PCR were designed using the Primer 6.0 Software. qRT-PCR was performed in a Mini Option real-time detector (Roche Light Cycle@480). The qRT-PCR reaction solution included 5 μl Talent qPCR Premix (2×) (TIANGEN Biotech Co., Ltd., Beijing), 0.4 μl PCR forward primer (10 μM), 0.4 μl PCR reverse primer (10 μM), 2.0 μl cDNA solution (20 ng), and 2.2 μl RNase-free water. The reaction conditions were followed by the recommendation of the instruction. Agarose gel electrophoresis was performed on all amplifiers to determine their size. The expression level of each gene was normalized towards the reference gene (18 s rRNA). The optimized comparative Ct (2^-ΔΔCt) value method was applied here to calculate gene expression levels.

To validate the transcriptome sequencing results, three genes with AS events were selected for RT-PCR and four genes from differential expressed gene analysis were selected for qRT-PCR. To summarize, total RNAs were used to synthesize the cDNA firstly using the GoScript™ Reverse Transcription System (Madison, WI, USA, Promega). Primer pairs of these nine genes were designed using Primer Premier 6.0 Software (Table S1). For validation of AS events, the reverse transcription products of three genes were subjected to PCR analysis to obtain PCR products for agarose gel electrophoresis. To validate the differential expressed genes between NH and DH, the qRT-PCR of four genes were performed according to the manufacturer’s protocol in a LightCycler^® 480 system (Indianapolis, IN, USA, Roche Applied Science) using a miRcute Plus miRNA qPCR Kit (SYBR Green) (TIANGEN Biotech, Beijing, China) and Talent qPCR Premix (SYBR Green) kit (TIANGEN Biotech, Beijing, China). In this process, 18S rRNA was used as the internal control (reference genes), each gene was amplified in three biological replicates and three technical replicates, relative fold-change was calculated using the 2^−∆∆CT method [38 (link)], and a student’s t-test was used to determine the statistical significance (p < 0.05) using R software.

Twelve differentially expressed unigenes were randomly selected and quantitatively analyzed using RT-qPCR to verify RNA-seq results. Primers were designed using Primer Premier 5.0 (Supplementary Table S2). The Talent qPCR PreMix (SYBR Green) Kit (Tiangen, Beijing, China) was used for triplicate RT-qPCR reactions with a CFX96 Touch Real-Time PCR System (Bio-Rad, Hercules, CA, USA) based on provided directions. The cycle threshold (Ct) and 2^−ΔΔCt method were utilized to assess relative transcript levels for each gene, which were normalized using UBC as internal controls.

Total RNA was extracted using TRIzol (Invitrogen, USA). RNA was reverse transcribed using a Quantscript RT Kit (TIANGEN Biotech, China). RT-qPCR was performed using a Talent qPCR PreMix (SYBR Green) Kit (TIANGEN Biotech, China) on a Bio-Rad machine using the following conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 s, annealing and extension at 60°C for 15 s, 40 cycles. Gene expression was measured using the 2^-ΔΔCt formula and the mRNA level of Actin as the control.