Trieasy total rna extraction reagent

Four cancerous tissues and 4 normal tissues were collected from the Hospital of Stomatology, Jilin University. TRIeasy Total RNA Extraction reagent (Yeasen Biotech, Shanghai, China) was added to the tissue and homogenized with a homogenizer (JXFSTPRP-24, Shanghai Jingxin Industrial Development Co., Ltd., Shanghai, China) using the following parameters: 3 grinding times, a frequency of 73 Hz, an interruption time of 3 s, and a running time of 60 s. The cDNA was synthesized using the Hifair III 1st strand cDNA Synthesis SuperMix for quantitative real-time PCR (Yeasen Biotech). The primer sequences used are shown in Table 1.

Total RNA was extracted from spleen, the J-Pd and the ileum tissues with TRIeasy™ Total RNA Extraction Reagent (10606ES60, YEASEN Biotech Co. Ltd, Shanghai, China). The first-strand complementary DNA (cDNA) was synthesized using ‘Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus)’ (11141ES10, YEASEN Biotech Co. Ltd, Shanghai, China). Quantitative real-time qPCR (qRT-PCR) analysis was performed on a real-time PCR system according to ‘Hieff UNICON® Power qPCR SYBR Green Master Mix (Antibody technique, Low Rox)’ (11202ES08, YEASEN Biotech Co. Ltd, Shanghai, China). mRNA content was calculated by 2^−ΔΔCt relative quantification with β-actin as an internal reference. All mRNA primer sequences were shown in Table 1. All primers were designed and synthesized by Accurate Biology Co., Ltd, Hunan, China.

Total RNA from B. striata tissues was isolated using the TaKaRa MiniBEST Plant RNA Extraction Kit (No. 9769, TaKaRa, Japan), and total RNA from the basal third of the mature inflorescence stems of A. thaliana was isolated using the TRIeasy™ Total RNA Extraction Reagent (10606ES60*, Yeasen, China). Approximately 100 mg of tissue powder ground with liquid nitrogen was used to extract total RNA, and then the RNA solution was stored at −80°C. Two micrograms of total RNA was reverse-transcribed into cDNA using SMART MMLV Reverse Transcriptase (No. 639523, Clontech) according to the manufacturer’s protocol. In brief, 2 μg total RNA was adjusted to 11 μl with RNA-free H₂O, 1 μl Oligo(15 T) was added and incubation was performed at 70°C for 3 min. After cooling on ice, 4 μl of 5 × first-strand buffer, 2 μl of Advantage UltraPure PCR Deoxynucleotide Mix (No. 639125, Clontech), and 2 μl of 10 mM DTT were added in sequence, and then the preparation was incubated at 42°C for 60 min to obtain 20 μl of cDNA solution. The cDNA solution was stored at −20°C.

The total RNA was extracted by adding 1 mL of lysis solution (TRIeasy™ Total RNA Extraction Reagent, Yeasen, Shanghai, China) to the cell samples (approximately 1 × 10⁶ cells per sample). A two-step triple pre-mix kit, MonScript™ RTIII Super Mix with dsDNase (Two-Step, Monad, Suzhou, China), was used to remove genomic DNA contamination from the total RNA and synthesize the first-strand cDNA. The reaction system was prepared using a pre-mixed solution for real-time PCR amplification (HieffTM qPCR SYBR Green Master Mix, Yeasen, Shanghai, China). The expression of MSH2 in the cells was detected via qRT-PCR using the primer msh2i-iden-F1/R1, and the internal reference was 17SrRNA.

According to the manufacturer’s protocol, total RNA was extracted using TRIeasy™ Total RNA Extraction Reagent (10606ES60, Yeasen, Shanghai, CN), and RNA concentration and purity were determined by Nano Drop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The RNA purity was considered as qualified when A₂₆₀/A₂₈₀ was between 1.8 and 2.0. The procedures about reverse transcription (11141ES10, Yeasen, Shanghai, CN) and quantitative polymerase chain reaction (qPCR, 11198ES08, Yeasen, Shanghai, CN) were detailed in the kit instructions. The qPCR reaction was performed by real-time fluorescent quantitative PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The primer sequences involved in this study can be found in Additional file 1: Table S1, and the transcription level of each gene was standardized by the internal control gene, ACTB, which is encoding for β-actin.

Total RNA from MC‐3T3 cells was extracted using TRIeasy Total RNA Extraction Reagent, provided by Yeasen Biotechnology in Shanghai, China. This RNA was then converted to cDNA using the Hifair III First‐Strand cDNA Synthesis SuperMix, suitable for quantitative polymerase chain reaction (qPCR) with gDNA digester plus (also from Yeasen). The RT‐qPCR was performed using a CFX96 RT‐PCR Detection System from Bio‐Rad, CA, USA, and the Hieff qPCR SYBR Green Master Mix (Low ROX Plus) by Yeasen. Primer sequences are listed in Table 1. The qPCR cycling protocol was in accordance with the method described by Li et al. (2021). Relative mRNA expression levels were quantified using the comparative 2^−ΔΔCt method and normalized against the internal control gene, Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH).