Hiscript reverse transcriptase

Total RNA was extracted from the regenerated bladder tissue using TRIzol reagent (Invitrogen, Thermo Fisher Scientific Inc., NY, USA). cDNA was synthesized by reverse transcription of total RNA using the Hiscript Reverse Transcriptase (Vazyme Biotech Co. Ltd., Nanjing, China), according to the manufacturer’s instructions. cDNA products were diluted 10 times, 4 μl was used as the templates for qRT-PCR. The transcription levels of the genes for VEGF, VEGFR2, SDF-1α, and CXCR4 were determined by qRT-PCR analysis with an Eco™ Real-Time PCR System (ABI7900, Illumina, Inc., CA, USA) using Power SYBR Green PCR master mix (2×) (Vazyme Biotech Co. Ltd., Nanjing, China). qRT-PCR was performed with a protocol of 50 °C for 2 min, 95 °C for 10 min, 40 cycles at 95 °C for 30 secs and 60 °C for 30 sec. Target mRNA expression levels were normalized to the housekeeping genes of rat β-actin as an internal control for quantification by 2^-(∆∆Ct). The primers used for the qRT-PCR analysis are listed in Table 3. Each assay was performed in triplicate.

Total RNA was extracted from plant tissue by using the EASY spin plant RNA extraction kit (Aidlab, Beijing, China) according to the manufacturer’s instructions, including the on-column DNase treatment. RNA was quantified using a Nanodrop 1000 (Thermo Fisher Scientific) and cDNA synthesized using Hiscript Reverse Transcriptase (Vazyme, Nanjin, China) and oligo(dT) primers (Vazyme). qRT-PCR was performed using Bio-Rad SYBR Green Supermix, and PCR parameters were as follows: 95 °C for 5 min (first cycle); 40 cycles of 95 °C for 10 s, 58 °C for 10 s, and 72 °C for 30 s; and a final cycle of 72 °C for 5 min. PCRs were performed in triplicate with a Bio-Rad CFX Connect^TM Real-Time Detection System (Bio-Rad, Hercules, CA, USA). Gene expression levels were calculated by a comparative ΔΔCt method as described in the manufacturer’s instructions for the CFX Connect^TM Real-Time Detection System. All primers, including internal controls used for measurement of transcript abundance, are shown in Supplementary Table S1.

Tissue biopsies obtained from HCC patient were weighted and homogenized by sonication in Qiazol Lysis Reagent (Qiagen, Hilden, Germany). RNA was isolated using chloroform extraction and dissolved in the TE buffer. The quantity and purity of RNA was evaluated using Nanodrop 2000 (ThermoFisher Scientific Inc., USA), and 1 μg of total RNA was reversely transcribed using HiScript Reverse Transcriptase (Vazyme Biotech Co. Ltd, China). qRT-PCR was performed in LightCycler 480 (Roche Diagnostics Ltd, Germany) using AceQ qPCR SYBR Green MasterMix (Vazyme Biotech Co. Ltd, China). Primers for the targeting gene CFHR4 and housekeeping gene ACTB were listed below. Results were analyzed using dCt method.

Total RNA was extracted from intestinal tissues using TRIzol reagent (Invitrogen, Waltham, MA, USA). The amount of RNA was measured by NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Purified RNA was reversed into cDNA by HiScript^® Reverse Transcriptase (Vazyme Biotech, Nanjing, China) and then immediately stored at −20 °C for qRT-PCR. Expression of zonula occludens-1 (ZO-1), apoptosis- (Caspase3, Caspase8, p53, Bcl-2 and Bax) and immune-related (MyD88, ALF1, Relish and Dorsal) genes was measured. Hieff^® qPCR SYBR^® Green (Yeasen Biotechnology, Shanghai, China) and a Roche LightCycler96 were used for qRT-PCR detection. EF1-α was used as an internal reference gene. Melting curves were generated to investigate the specificity of each amplified products. Relative gene expression was calculated by 2^−ΔΔCT method [54 (link)]. All samples were run in triplicates. Detailed primer sequences are presented in Table A1.

RAW264.7 cells (2×10⁶ cells/well) were incubated with lipopolysaccharide (LPS; 1 μg/ml) for 24 h and then treated with puerarin (50, 75, or 100 μM) for 24 h.
Total RNA was extracted with TRIzol^® Reagent (Thermo Scientific, Waltham, MA, USA), and reverse-transcribed with oligo-DT using HiScript™ Reverse Transcriptase (Vazyme, Beijing, China) according to manufacturer instructions. The primers used were synthesized by Genscript (Nanjing, China). The sequences were (forward and reverse, respectively) 5′-TTGGTCAGGTGAAGGGAGAC-3′ and 5′-GGATCACAGCCAGCTTTCAG-3′ for NF-κBIA; 5′-TTGCGTGAAGGCTTGAGATG-3′ and 5′-CTGGACAGGATGGAGGGTTT-3′ for BCL2; 5′-AAGCCTTCTCCAACCTCTCC-3′ and 5′-GCTGGGCAAAGAATGCAAAC-3′ for PTGS2; 5′-AACGGATTTGGCCGTATTGG-3′ and 5′-CATTCTCGGCCTTGACTGTG-3′ for the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
qRT-PCR was done using SYBR™ Green Master Mix (Vazyme) in the QuantStudio 6 Flex system (Applied Biosystems, Foster City, CA, USA). The PCR cycling profile was: one cycle at 50°C for 2 min and 95°C for 10 min, 40 cycles at 95°C and 60°C for 30s. Fluorescence signals were detected using the QuantStudio 6 Flex system. Gene-expression data were normalized to that of the endogenous control GAPDH. The 2^−ΔΔCT method was the basis for relative expression of genes.