Universal sybr qpcr master mix

Field-fresh tissue samples were collected, ground in liquid nitrogen, and used for total RNA extraction using the EASY Spin Plant RNA Rapid Extraction Kit (RN09, Aidlab, Beijing, China). A total of 1 μg of total RNA was used for genomic DNA removal and first-strand cDNA synthesis using the HiScript II Q RT SuperMix for qRT-PCR Kit (R223, Vazyme). The qRT-PCR reaction mixture (20 μL) included 10 μL of Universal SYBR qPCR Master Mix (Q711, Vazyme), 5 μL of diluted cDNA, 0.5 μL of 10 μmol/L forward and reverse primers each, and 4 μL of ddH₂O. Reactions were induced in triplicate, with fluorescence detection enabled via a Bio-Rad CFX Connect Real-Time PCR System, using the ΔΔCq method. GhHistone3 (Accession number: AF024716) served as the reference gene, with specific primer sequences provided in the Supplementary Materials.

A total RNA isolation kit was purchased from Vazyme (RC101). For RT-qPCR, RNA was reverse transcribed to cDNA by reverse transcriptase (Vazyme, R312-01). qPCR analyses were performed with Universal SYBR qPCR Master Mix (Vazyme, Q711-02). For the results analysis, GAPDH was used as a reference. The primers are listed below: MRE11A: Fwd 5′-ATCGGCCTGTCCAGTTTGAAA-3′ and Rev 5′-TGCCATCTTGATAGTTCACCCAT-3′. PMS2: Fwd 5′-TTTGCCGACCTAACTCAGGTT-3′ and Rev 5′-CGATGCGTGGCAGGTAGAA-3′. GAPDH: Fwd 5′-GGAGCGAGATCCCTCCAAAAT-3′ and Rev 5′-GGCTGTTGTCATACTTCTCATGG-3′.

The total RNA was isolated from the exosomes and cells by using TRIzol reagent (Life Technologies, USA). For miRNA, the first-strand cDNA was synthesized by using miRNA 1st Strand cDNA Synthesis Kit (Vazyme, China), then real-time PCR was performed by miRNA Universal SYBR qPCR Master Mix (Vazyme, China) under LightCycler480 Real-Time PCR Detection System (Roche, Switzerland). For mRNA, the first-strand cDNA was synthesized by using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, China), then real-time PCR was performed by Universal SYBR qPCR Master Mix (Vazyme, China). U6 was used as an internal reference for miRNAs, and beta-actin was for mRNAs. Comparative quantification was determined using the 2^−ΔΔCt method. The primer sequences used are summarized in Supplementary Table S1.

Total RNA was isolated from liver tissues using the TRIzol Total RNA Isolation Kit (B511321; Sangon Biotech Co., Ltd., Shanghai, China). According to the manufacturer’s instructions, the samples were diluted using Trizol, and total RNA was obtained after homogenization, separation, extraction, purification, adsorption, and elution. Next, we measured the concentration of the collected RNA, and a reverse transcription system was used to convert 1 μg of RNA into cDNA (R323; Vazyme Biotech Co., Ltd., Nanjing, China). Real-time PCR was performed using a BioRad-CFX384 Touch thermocycler (Bio-Rad, Hercules, CA, USA) and Universal SYBR qPCR Master Mix (Q711; Vazyme Biotech Co., Ltd., Nanjing, China). All samples were tested in quadruplicate. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene and the data were standardized using the ΔΔCT method for each sample. The target genes included the lipid metabolism genes fatty acid synthase (FASN), acylglycerol lipase (MGL), hormone-sensitive lipase (HSL), and patatin-like phospholipase domain-containing protein 2 (ATGL).

For qPCR, total RNA was extracted using TRIzol reagent (Invitrogen) and cDNA was synthesized using HiScript III RT SuperMix (Vazyme, Nanjing, China). The reaction was performed using the universal SYBR qPCR Master Mix (Vazyme). IB, IF and IHC were performed following conventional protocols. IHC scores were calculated as we described previously [24 (link)]. Besides IB, METTL3, ALKBH5, METTL14, WTAP, YTHDF1, ENO1 and FTO were also measured using ELISA kits from Lichen Biotech (Shanghai, China). The qPCR primer sequences and antibody information for IB, IF and IHC are listed in Supplementary Tables 5-6.