Revertaid first strand cdna synthesis kit

Total RNAs were extracted with the TRIzol reagent (Cat No. 15596018, Invitrogen, USA) according to the manufacturer’s instructions. The first strand complementary DNAs (cDNAs) were then synthesized using the Revert Aid First Strand cDNA Synthesis Kit (R233-01, Vazyme, Nanjing, China), and quantitative PCR (qPCR) was carried out with the Power SYBR Green PCR Master Mix (Cat No. 4367659, ABI, Life Tech, Carlsbad, CA, USA) on an ABI 7900 HT Fast Real-Time PCR System. For each gene of interest, three biological and technical repeats were performed, respectively, and the relative expression level was calculated according to the 2^−∆∆CT algorithm [72 (link)]. The primers used for these quantitative assays are provided in Table 1.

Total cellular RNA was isolated from cells or tissues using TRIzol reagent (Vazyme, Jiangsu, China) and RNA was reverse transcribed using a Revert Aid First Strand cDNA Synthesis Kit (Vazyme). qPCR was performed using SYBR GREEN master mix (Vazyme) on a Bio-Rad CFX-96 fluorescence quantitative PCR instrument. mRNA levels of the genes of interest were standardized with an internal control (18s rRNA). Primer sequences are provided in Table 1.

Total RNA from the colon tissue was extracted, and cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd.; Nanjing, China). Gene expressions of Mucin-2, occluding, and sodium-hydrogen exchange protein (NHE)3 were detected using real-time quantitative polymerase chain reaction [29 (link)]. mRNA expression was determined using a real-time quantitative PCR instrument (CFX Connect; Bio-Rad), as well as a universal iTaq SYBR green Supermix and associated primers (Table 3). The conditions were 40 cycles of 95 °C for the 30 s, 95 °C for 5 s, and 60 °C for 30 s. The β-actin gene was used as a reference gene for the calculation of quantitative expression of the target gene. The expression of genes was calculated using the 2^−ΔΔCt method with the control group as the base.

The total RNA was extracted using RNAiso Plus (Takara, China) according to the manufacturer’s protocol. The complementary DNA (cDNA) synthesis was carried out with RevertAid First Strand cDNA Synthesis Kit (Vazyme Biotechnology, Nanjing, China) according to the manufacturer’s instructions. Primer Premier 5.0 software was used to design primers, and rpoB was selected as the internal control gene (Supplementary Table S1). Reactions were performed using ChamQ SYBR qPCR Master Mix (Vazyme Biotechnology, Nanjing, China) in a 20 μl final volume containing 10 μl SYBR premix Ex Taq II, 0.8 μl of each primer, 0.4 μl ROX Reference Dye, 2 μl of diluted cDNA, and 6 μl sterile distilled water. Quantitative real-time RT-PCR (qRT-PCR) was performed on a StepOne™ Real-Time PCR System (48-well format; Applied Biosystem, United States) with the following program: 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, at 60°C for 1 min. A melting curve analysis was performed to confirm product specificity. The relative expression levels were calculated using the 2^-ΔΔCT method. The FC in mRNA level were determined using the −ΔΔCt data analysis method. All samples were analyzed in three replications. The primers used in this study were listed in Supplementary Table S1.

Total RNA was isolated from human GBM cells using RNAiso Plus (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. Real-time RT-PCR was conducted using the RevertAid First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and the SYBR GREEN PCR Kit (Vazyme, Nanjing, China) following the manufacturer's instructions. The relative expression of the various genes was normalized to β-actin. The following sequences were used for the qRT-PCR primers, and the results were evaluated using the standard ΔΔCt method.

Total RNA isolation from the colon tissue was performed using a FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., Nanjing, China), and then a RevertAid First Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., Nanjing, China) was used for cDNA synthesis. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed using β-actin as an internal control to identify the expressions of mucin2 (MUC2), ZO-1, claudin-1, and occludin [41 (link)]. RT-qPCR was carried out on a CFX96 Real-Time System (Bio-Rad, Hercules, CA) using SYBR Green super mix (Bio-Rad, Hercules, CA) under the following program: 2 min at 95°C, 39 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. The 2^-∆∆Cq method was used to analyze the results. Table 1 lists the sequences of primers used in this study.

To verify the sequencing results, three DEGs and two DELs were selected for further confirmation by RT-qPCR method. PCR primers specific to the selected genes and the reference gene (18S) were designed using Primer Premier 5.0 (Applied Biosystems). Total RNA was reverse-transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Vazyme, Nanjing, China, R211-01). RT-qPCR for mRNA detection was carried out in Roche LightCyler 480 system (Roche, Mannheinm, Germany) with SYBR Green (Vazyme, Nanjing, China, Q711) according to the manufacturer’s instructions, The RT-qPCR data were analyzed using the 2^−ΔΔCT method, as previously described [26 (link)]. In brief, the relative fold changes of mRNA expression were quantified by normalizing the cycle threshold (CT) value of the experimental gene to the mean CT value of the reference gene 18S.