Quantstudio 5

Gene expressions of C3 were determined by qRT-PCR. Total RNA was isolated from the lung tissues of mouse and human COPD patients, and the cultured cells were treated with Trizol reagent (Thermo Fisher Scientific, MA, USA) and the Nano Drop 2000 was used to measure RNA concentration. RNA was then reverse transcribed into cDNA using the HiScript III RT Supermix for qPCR (+gDNA wiper) Kit (R323-01, Vazyme, Nanjing, China). The qPCR reactions were performed on the Applied Biosystems^® QuantStudio^® 5 in a 20 μl reaction system using the ChamQ Universal SYBR qPCR Master Mix Kit (Q711-02, Vazyme, Nanjing, China).

Total RNA was extracted from cell tissues using a TRIzol reagent kit (Invitrogen). PCR was amplified and sequenced using Illumina NovaSeq 6000 by Gene Denovo Biotechnology (Guangzhou, China). We identified genes as significantly differentially expressed if they had a fold change ≥1 and a false discovery rate <0.05. Total RNA was isolated from osteosarcoma cell lines using the Total RNA Kit (R6834-01, Omega). One microgram of RNA was used for cDNA synthesis using a High-Capacity cDNA Reverse Transcription Kit (Q711-02, Vazyme). Transcript levels were determined on an Applied Biosystems QuantStudio 5 real-time PCR system using the Vazyme iTaq Universal SYBR Green Supermix (R223-01, Vazyme). Relative gene expression was calculated using the 2 -∆∆Ct method. The primer sequences are listed in Supplementary Table 2.

Serum total RNA was extracted using a rapid blood total RNA extraction kit (BioTeke Corporation, China), while total RNA from tissues and cells was extracted using a TRIzol reagent (Invitrogen, Germany). cDNA was produced using the RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific, USA), and quantitative real-time PCR (qRT-PCR) was performed on ABI QuantStudio 5 with the 20 μL reaction system, which included 10 μL ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., China), 1 μL primers, 3 μL enzyme-free water, and 5 μL cDNA. U6 was used as an internal reference, and the expression of tRF-17-WS7K092 was calculated by the 2^−∆∆CT method. All primers used in the study were synthesized by RiboBio (Guangzhou, China).