Hifair 2 1st strand cdna synthesis kit

After obtaining the hub gene, we verified the hub gene by quantitative real-time polymerase chain reaction (qPCR). RNAiso Plus (9,109; Takara, Dalian, China) was used to extract total RNA, and 1 μg of total RNA was then transcribed into complementary DNA by using the Hifair^® Ⅱ 1st Strand cDNA Synthesis Kit (11119ES60; Yeasen Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Use this system for mRNA amplification: 85°C for 5 min, then 40 cycles of 95°C for 10 s and 60°C for 30 s. ACTB was used as an internal control for mRNA evaluation. To standardize SOX9 gene expression, ACTB expression levels were assessed as housekeeping genes, and comparative CT (2^−ΔΔCt) methods were used for the analysis. 2^−ΔΔCt indicates the ratio of target gene expression between cancer and normal groups. ΔΔCT = ΔCt cancer group - ΔCt normal group and ΔCt = Ct target gene - Ct control gene. Table1 shows the primers of SOX9 and ACTB.

Circulating neutrophils were obtained from the blood sampled before rtPA treatment in all patients, preserved in TRIzol Reagent (Invitrogen, Cat#15596026) and then stored in the −80°C refrigerator until detection. All samples were handled according to the same protocol throughout the study.
Total RNA was extracted from the neutrophil samples with TRIzol Reagent, and then quantified with nanodrop. According to the manufacturer’s instructions, 1 μg of total RNA in a final volume of 20 μl was used for miRNA First Strand cDNA Synthesis (Tailing Reaction) (Sangon Biotech, Cat#B532451) and lncRNA reverse transcription was conducted with a Hifair^® Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China, Cat#11121ES60). Total cDNA was followingly prepared for RT-qPCR with the Hieff^® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China, Cat#11202ES08) in a QuantStudio^™ Real-time PCR System (Applied Biosystems, United States). The expression of universal U6 was used as the control for miRNA, and GAPDH was used as the control for lncRNA. 2^−ΔΔCT method was used for relative quantification of noncoding RNA expression. Each quantitative PCR assay was performed in triplicate independently. The sequences of the primers used in the present study were listed in file e1. All samples were tested by a professional technician blinded to the clinical assessments.

Total RNA was isolated from rat renal cortex or GMCs with TRIzol, and the RNA was reverse transcribed into cDNA by Hifair® Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen, China), HiScript Reverse Transcriptase or HiScript II Q RT SuperMix (Vazyme, China). Regular PCR was done with 2 × Taq Master Mix (Vazyme). The data were normalized to GAPDH. The qPCR amplification was performed with AceQ qPCR SYBR Green Master Mix (Vazyme) or Hieff® qPCR SYBR Green Master Mix (Yeasen) in an ABI StepOnePlus. The data were normalized to β-actin, and relative gene expression was calculated using the 2^-ΔΔCT method. Relative primer sequences are provided in Table S1.

Reverse transcription (RT) reactions were carried out using the Hifair^® Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology CO., Ltd., Shanghai, China) following the standard protocol. The qRT-PCR test was conducted by using SYBR Green Premix ExTaq (Takara, Dalian, China) on the ABI 7500system (Applied Biosystems, Carlsbad, CA, USA). The relative mRNA expression of related genes was calculated by reference to β-actin using the 2^−ΔΔCt method [21 (link)]. The primers were designed using Primer Premier 5.0 software.

We used RNA-easyTM Isolation Reagent (Vazyme, Nanjing, China) to extract RNA from sperm samples according to the manufacturer’s instructions. And cDNA was synthesized with the Hifair^® Ⅱ 1st Strand cDNA Synthesis Kit (Yeasen, Shanghai, China). The detailed procedures of quantitative real-time polymerase chain reaction (qRT-PCR) were described previously (Chen et al., 2021 (link)). Then, primers were synthesized (Tsingke, Wuhan, China), and sequence information is shown in Supplementary Table S3. The 2^−ΔΔct relative quantification algorithm was utilized to evaluate the mRNA levels of hub genes in each sample.