Gdna clean for qpcr 2

The total RNA isolated from the vaginal epithelium was subjected to quantitative real-time PCR (qRT-PCR). Briefly, we synthesized cDNA using the Evo M-MLV RT kit with gDNA Clean for qPCR II (Accurate Biology, China). We then performed qRT-PCR employing the SYBR Green premix Pro Taq HS qPCR kit (Accurate Biology, China) following the manufacturer’s protocol. We adopted the following qRT-PCR protocol: 95°C for 10 min, 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 20 s. Primers were determined using NCBI Primer-BLAST, and all reactions were repeated in triplicate (see Table S7 at https://zenodo.org/record/7537183). The relative expression of genes was calculated via the 2^−ΔΔCT method and normalized using β-actin (ACTB).

SteadyPure Universal RNA Extraction Kit (Accurate Biology) was used to isolate total RNA, and the first-strand cDNA was synthesized by using the Evo M-MLV kit with gDNA clean for qPCR II (Accurate Biology). The specific primers of HvZF-HD and HvActin genes were showed in Table S1 [26 (link)]. Real-time PCR experiments were conducted using TransTaq-T DNA Polymerase (TransGen, Beijing, China) under the following cycling program: initial denaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s. Real-time PCR experiments were performed in three independent biological replicates and three technical replications to determine the average Ct values. The relative expression levels of HvZF-HD genes were calculated by 2^−∆∆CT method [27 (link)].

The total RNA was extracted from PBMCs by using SparkZol Reagent (SparkJade, Shandong, China) and then reverse transcribed into cDNA by using an Evo M-MLV RT Kit containing gDNA Clean for qPCR II (Accurate Biology, (Hunan), Co., Ltd.). The expression levels of lncRNAs and mRNAs were detected by RT-PCR, which was performed by using a SYBR Green qPCR Mix kit (With ROX) (SparkJade, Shandong, China). The conditions used for qPCR were as follows: 95°C for 2 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s. The total RNA was reverse transcribed into miRcDNA by using a miRNA first Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, Nanjing, China). miRNA expression was achieved by performing RT-PCR with miRNA universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) under the following conditions: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The primers used for amplification of lncRNAs, miRNAs, and mRNAs are shown in Table 1. Each RT-qPCR analysis was repeated more than three times. Relative levels of gene expression were calculated by using the 2^−ΔΔCT method (Ayuk et al., 2016 (link)).