Hiscript rt supermix for qpcr gdna wiper

The total RNA of jejunum tissue was extracted by RNA-easy™ Isolation Reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions, and then the concentration was determined using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, United States), RNA purity was verified by the ratio of absorbance at 260 nm/280 nm. Next, the total RNA was reverse-transcribed into complementary DNA (cDNA) by HiScript ^® RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, China) using the Bio-Rad DNA Engine (United States). The quantitative real-time polymerase chain reaction (RT-PCR) analysis was performed on CFX-96 Real-Time PCR System (Bio-Rad, United States) using ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China). To construct a 10-μl reaction system, including 1 μl of cDNA template, 1 μl of reverse primer (4 μM), 1 μl of forward primer (4 μM), 2 μl of dd H₂O and 5 μl of ChamQ SYBR Color qPCR Master Mix (2×). Primers of the target gene were designed with Primer 5.0 (Premier Biosoft International, Palo Alto, CA, United States) and listed in Table 2. All designed primers synthesized by GENEWIZ Biotechnology Co., Ltd. (Suzhou, China). β-actin were selected as the reference genes, and the relative mRNA abundance of target gene mRNA was calculated using the method of 2^–ΔΔCt, as described by Livak and Schmittgen (2001) (link).

Total RNA isolated from brains was transferred to cDNA using HiScript RT SuperMix for qPCR (+gDNA wiper) purchased from Vazyme (Nanjing, China). For gene expression analysis, PCR amplification was performed using Taq Pro Universal SYBR qPCR Master Mix purchased from Vazyme and the samples were run on a CFX96™ Real-Time PCR Detection system (Bio-Rad, v2.2). The primers purchased from Invitrogen were designed using PrimerBank and Primer-BLAST (Supplementary Table S1). To calculate gene expression, the 2^−ΔΔCt method was used with GAPDH expression as a normalizer and an untreated sample as relative control.

The HiScript RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) was applied to reverse transcribe the total RNAs of each group following the protocol. QPCR using the SYBR Green kit (TaKaRa, Dalian, China) was applied to evaluate the expression levels of the circRNAs and mRNAs. Primers for mRNAs were routinely used. To amplify the circRNA junctions, we designed specific divergent primers based on the sequence obtained from the database “circBase” (http://www.circbase.org/). By using the 2-ΔΔCt method that normalizes against the expression of the β-actin gene, the circRNA and mRNA relative expressions were calculated. GenePharma (Shanghai, China) was applied to design, verify, and synthesize all primers. All sequences are as in Table 1.