Abi quantstudio6 q6

For q-PCR validation of tsRNA, the total RNA was extracted by TRIzol Reagent (Takara Biotechnology) from plasma exosomes, BMSCs exosomes and HUVECs, respectively. For mRNA, the total RNA was isolated from HUVECs. PrimeScriptTM RT kit and gDNA Eraser kit (TaKaRa, Tokyo, Japan) were utilized to reverse transcribe RNA into cDNA. SYBR Green PCR kit and ABI QuantStudio6 Q6 (Applied Biosystems, Foster City, CA) were applied to test the expression of tsRNA and mRNA, according to the manufacturer’s instruction. Response conditioning was set according to the following procedure: predenaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s, and annealing at 60 °C for 1 min. U6 (for tsRNA in exosomes and HUVECs) and GAPDH (for mRNA in HUVECs) were the internal parameters of q-PCR (Cheng et al., 2020 (link); Han et al., 2021 (link); Sun et al., 2021 (link); Qu et al., 2022 (link)). Three independent experiments were performed. Quantitative analysis was performed by 2^−ΔΔCt method. The specific PCR primers are listed in Table S1.

Total RNA was extracted from the cultured cells and fresh frozen cervical tissues using Trizol reagent (Vazyme Biotech, Nanjing, China), according to the manufacturer’s instructions. Reverse transcriptase reactions were performed according to the manufacturer’s protocol using HiScript II Q Select RT SuperMix as the qPCR reverse transcriptase reagent (Vazyme Biotech, Nan Jing, China). Gene expression levels were normalized to the house-keeping gene β-actin. Reactions were performed in triplicate with ABI QuantStudio6 Q6 (Applied Biosystems, USA). Primer sequences are listed in Supplementary Table S5.