Transzol up kit

Fifty to two hundred nanograms of total RNA was extracted from mouse kidney using a TransZol Up Kit (ET111-01, TransGen Biotech, Beijing, China). The quantity and concentration of RNA were evaluated by 1.2% agarose gel electrophoresis and Nanodrop 2000 (Thermo Fisher, USA). The total RNA was transcribed into cDNA utilizing a High Capacity cDNA Archive Kit (Applied Biosystems, CA). Primers for the evaluated genes are listed in Table 1. Quantitative real-time RT-PCR (qRT-PCR) was performed in 96-well plates (0.5 μL (10 μM) forward primer, 0.5 μL (10 μM) reverse primer, 1 μL template cDNA (cDNA, 10–15ng/μL), 10 μL Universal Master Mix, and 8 μL of RNAse-free water), all reagents were obtained from Applied Biosystems by Life Technologies, USA. All qRT-PCR reactions were performed at 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, and 62°C for 30 s, using two-step RT-PCR. All qRT-PCR reactions were performed on the ABI 7900 HT system and were conducted in triplicate to ensure methodological reproducibility.

We extracted 50–100 ng total RNA from mouse liver tissue using the TransZol Up Kit (ET111-01, TransGen Biotech, China). The quantity and concentration of RNA were evaluated in a 1.2% agarose gel and by Nanodrop 2000 (Thermofisher, Waltham, MA, USA). Total RNA was transcribed into cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Primers used to evaluate the genes phospholipase A2-3 (PLA2-3), receptor-interacting proteins-1 (RIPK-1), receptor-interacting proteins-3 (RIPK-3), mixed lineage kinase domain-like protein (MLKL), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and GAPDH are indicated in Table 1, and GAPDH was used as the internal reference to assure equal loadings. Quantitative real-time RT-PCR (qRT-PCR) was performed using 96-well microwell plates in a total volume of 20 μL containing 1 μL template cDNA (10 ng/ μL), 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 10 μL of Universal Master Mix, and 8 μL of RNase-free water (Applied Biosystems, Foster, CA, USA). The qRT-PCR reactions were performed at 94 °C for 30 s, followed by 40 cycles at 94 °C for 5 s and 62 °C for 30 s by using two-step RT-PCR. All qRT-PCR reactions were performed in the ABI 7900 HT system and in triplicate to ensure methodological reproducibility.

Total RNA was extracted from the tissues using TransZol Up kit (Transgen, Beijing, China). Use Easyscript kit with gDNA Remover (Transgen, Beijing, China) to reverse transcribe the extracted RNA into cDNA, and then use Green qPCR Supermix kit (Transgen) to perform qPCR in real-time fluorescence quantitative PCR Detection System (Applied Biosystems, 7900, United States). PCR primers are synthesized by Sangon (Shanghai, China), using to obtain the expression level of mRNA. The primers (5′-3′) used were listed as follows: AR (mice) forward primer: TCC​AAG​ACC​TAT​CGA​GGA​GCG, reverse primer: GTG​GGC​TTG​AGG​AGA​ACC​AT; PDCD4 (mice) forward primer: AAA​GAC​GAC​TGC​GGA​AAA​ATT​CA, reverse primer: CTT​CTA​ACC​GCT​TCA​CTT​CCA​TT; GAPDH (mice) forward primer: AGG​TCG​GTG​TGA​ACG​GAT​TTG, reverse primer: TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA; U6 (mice) forward primer: CTCGCTTCGGCAGCACA, reverse primer: AAC​GCT​TCA​CGA​ATT​TGC​GT. For the detection of miR-21 expression, use the mmu-miR-21a-5p bulge-loop RT primer designed by RiboBio (Guangzhou, China) for reverse transcription, and then use the mmu-miR-21a-5p specific primer pair for qPCR. MiRNA uses U6 as an internal reference, and mRNA uses GADPH as an internal reference. Use the 2-ΔΔCt value to quantify the fold change and determine relative gene expression.

The total RNA was isolated from the seedlings of C. acuminata by using the TransZol UP Kit (Beijing TransGen Biotech Co., Ltd., China), according to the manufacturer’s manual. Briefly, the seedlings were ground into fine powder within liquid nitrogen and the total RNA was extracted using the TransZol UP Kit. The RNA pellets were dissolved in diethylpyrocarbonate (DEPC)-treated water. The quality and quantity of the total RNA were determined by the ratio of OD₂₆₀ and OD₂₈₀ recorded from UV-1100D spectrophotometer (Shanghai Mapada Instruments Co., Ltd., China). The total RNA was stored at -80°C for further usage.

After the gonadal status of all fish had been determined via histology, six different stages of gonads were used to ascertain gene expression patterns. Extraction of total RNA followed the TransZol Up Kit (Transgene, ET111) manufacturer’s protocol. The quantity of total RNA was determined using a NanoDrop™ 1000 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The first-strand cDNA was synthesized from 1 μg total RNA with HiFiScript gDNA Removal RT MasterMix Kit (CWBIO, Beijing, China). cDNA synthesis was performed using the manufacturer’s protocol.

The total RNA of longan EC, ICpEC, GE, and EC treated with different hormones were extracted by TransZolUp kit (TransGen Biotech), and the total RNA of different development stages of the zygotic embryo was extracted by BioTeke kit (Cat. No. RP3301). The cDNA was synthesized with a Hifair^®III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen). Primer design was performed using Primer3 online software (https://primer3.ut.ee/cgi-bin/primer3/primer3web_results.cgi) (Supplementary Table 1). DlACTB, DlEF-la, and DlUBQ were used as internal control genes (Lin and Lai, 2010 (link)). The qPCR system was 20 μL, including HRbio™ qPCR SYBR^®Green Master Mix (No Rox) (Heruibio), 8.2 μL ddH₂O, 1 µL of 10-fold diluted cDNA, and 0.4 μL specific primer pairs, with three replicates. The reaction procedure was 95°C for 30 s, followed by 40 cycles of 95°C for 10 s and 58°C for 30 s, and 2^−ΔCT was used to calculate genes expression (Livak and Schmittgen, 2002 (link); Vandesompele et al., 2002 (link)). SPSS software was used for significant analysis of gene differences, and GraphPad 8.0.2 was used for the draft.

Total RNA of Day 10 nematodes treated as above was extracted using TransZol Up Kit (TransGen Biotech) and reverse‐transcribed with EasyScript® All‐in‐One First‐Strand cDNA Synthesis Kit (TransGen Biotech) according to the manufacturer's instructions. Quantitative real‐time PCR was carried out on a StepOnePlus Real‐Time PCR Instrument (Applied Biosystems) using PerfectStart^® Green qPCR SuperMix Kit (TransGen Biotech) with act‐1 as a reference gene. All reactions were performed in three technical replicates from three biological repeats. The primers of gene transcripts used for the PCR analysis are listed in Table S9.