Fastpure cell total rna isolation kit v2

HMGECs for mRNA extraction were seeded in 12-well plates at a density of 5 × 10⁴ cells/well. mRNA expression of TLR4, IL-1β, IL-6, IL-8, TNF-α, IL-12, IFN-γ, MMP-3, and MMP-9 (Table) was evaluated through quantitative RT-PCR (qRT-PCR). RNA was isolated using the FastPure Cell Total RNA Isolation Kit V2 (Vazyme, Nanjing, China) and qualified and quantified with Nanodrop (Thermo Scientific, Waltham, MA, USA). Then, 500 ng RNA was reverse transcribed with oligo dT and random primers as provided in the HiScript II Transcription Kit (Vazyme). qRT-PCR was performed with the SYBR green mix via the Lightcycler 96 system (LC96; Roche, Mannheim, Germany). The relative quantities of gene expressions were analyzed by using the comparative 2^−ΔΔCt method, and glyceraldehyde-3-phosphate dehydrogenase was characterized as the normalizing housekeeper gene.

Total RNA was extracted from selected cells using FastPure Cell Total RNA Isolation Kit V2 (RC112–01, Vazyme, Nanjing, China) following the manufacturer's instructions. Subsequently, HiScript II 1st Strand cDNA Synthesis Kit (R333–00-AC, Vazyme, Nanjing, China) was utilized to synthesize complementary DNA (cDNA) templates. The qRT-PCR was performed by using SYBR Green Master Mix (Q411–02, Vazyme, Nanjing, China). The primers are listed in Supplementary Table S2. Program execution and data acquisition was implemented with an qTOWER3G (analytic jena, German) sequence detection system. The internal reference for normalization of RT-qPCR results was GAPDH, and relative expression was calculated using the 2(-ΔΔCT) method. Each assay was conducted in triplicate. A primer with an amplification efficiency in the range of 90 % to 110 %, accompanied by a singular peak in the melting curve and a melting temperature (Tm) exceeding 80 °C, is considered to demonstrate robust amplification specificity and efficiency.