Transzol

Tibetan sheep were obtained from sheep farm (Xiangkemeiduo Sheep Industry Co. Ltd., Qinghai, China), and the experimental group included 433 ewes, which were selected randomly. The health and reproduction records of the animals were kept by the farmers. Their litter size was obtained from reproduction records. All efforts were made to minimize discomfort during the blood collection. Blood samples were collected from the jugular vein under the supervision of qualified veterinarians. Genomic DNA was extracted from blood sample of each sheep using an EasyPure Blood Genomic DNA Kit (TransGen Biotech, Beijing, China). Three ewes were selected from purebred herds of the same farm in Qinghai province. The three selected ewe (6 months old) were healthy, similar in weight, and pastured in similar conditions of grassland. After slaughtered, and the tissues from hypothalamus, hypophysis, heart, liver, spleen, lung, kidney, ovary, oviduct, uterus, rumen, duodenum, and longissimus dorsi were collected and immediately frozen in liquid nitrogen, and then stored at −80°C. The RNA of tissues was extracted by TransZol (TransGen Biotech). Total RNA for each tissue was reverse‐transcribed to cDNA by TransScript One‐Step gDNA Removal.

RNA from cultured cells or human tissue samples was prepared with TransZol (Trans Gen Biotech), and cDNA was synthesized from 5 µg of RNA using TransScript First-Strand cDNA synthesis SuperMix (Trans Gen Biotech). Gene expression was determined by real-time PCR using an iQTM SYBR Green SuperMix Kit (Bio-Rad, CA, USA) with a CFX96TM Real-Time system (Bio-Rad). All data were normalized to ACTB expression. The primers used are listed below. IL-6: forward primer 5’-GACAGCCACTCACCTCTTCA-3′, reverse primer 5’-AGTGC CTCTTTGCTGCTTTC-3’. MMP9: forward primer 5′-TTGACAGCGACAAGAAGTGG-3′, reverse primer 5′-GCCATTCACGTCGTCCTTAT-3′. SDHB: forward primer 5‘-ACAGCTCCC CGTATCAAGAAA-3’, and reverse primer 5‘-GCATGATCTTCGGAAGGTCAA-3’. ACTB: forward primer 5‘-TCCCTGGAGAAGAGCTACG-3’, and reverse primer 5‘-GTAGTTTCGTGG ATGCCACA-3’.

Total RNA, from tissue and bovine hepatocytes, was extracted with TransZol (TransGen Biotech, Beijing, China) according to the manufacturer’s protocols. RNA concentration and purity were determined using a Thermo Scientific NanoDropTM 2000 Spectrophotometers and agarose gel electrophoresis. The specific reverse transcribed primers for the miRNA were designed to construct miRNA cDNA libraries according to stem-loop method (Wang, 2009 (link)). MiRNA sequences were retrieved from miRBase database (https://mirbase.org/ftp.shtml, Dec. 12, 2020). cDNA was synthesized from 1 μg of RNA using a PrimeScript™ RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China). The cDNA was then used for quantitative real-time PCR (qPCR).

Total RNA was extracted from sweet orange leaves using TransZol (TransGen Biotech, Beijing, China) in accordance with the manufacturer’s instructions. One microgram of total RNA was used for first-strand complementary DNA synthesis using a Goldenstar RT6 cDNA Synthesis Kit (Tsingke Biotechnology, Beijing, China). All qRT-PCR reactions were run and analyzed using a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad, Munich, Germany) with a SYBR Green PCR Mastermix (Solarbio, Beijing, China). qRT-PCR was conducted following standard procedures and conditions as previously described (Peng et al., 2021b (link)). qRT-PCR gene-specific primers were designed using Oligo7 software (Supplementary Table 7).

Total RNA was extracted from cultures grown in TSB to an OD₆₀₀ of 3.0 using TransZol (Transgene, Canada) by following the manufacturer’s instructions. Residual DNA was removed using the Turbo DNase (Thermo Fisher, Canada). Reverse transcription was performed using the iScript kit (Bio-Rad, Canada). The expression of the hmqABCDEFG operon was determined by PCR targeting hmqA. The ndh gene served as a reference gene (Table S8) (72 (link)).

TransZol (TransGen, CN) was used to collect the total RNA from the cells, and RNA concentration and purity were determined with BioTek Epoch 2 microplate Reader (Biotek, USA), after which cDNA was synthesized via reverse transcription using Hifair ® II 1st strand cDNA Synthesis SuperMix (Yeasen, CN). cDNA was used for subsequent quantitative real-time polymerase chain reaction (YEASEN, CN). A two-step process was performed using the 2XRealStar Green Fast Mixture with ROX II (GenStar, CN), with actin serving as the internal reference. The relative gene expression levels were calculated using the 2^–△△Ct method. The primer sequences are listed in the Supplementary Material (Supplementary 1. Table S1).