Rna extraction kit

Total RNA was extracted using an RNA Extraction kit (Bioteck, Beijing, China). Single strand cDNA was reversely transcribed from 500 to 1000 ng RNA by using oligo dT-Adaptor primers and AMV reverse transcriptase kit (TaKaRa, Otsu, Japan). cDNA was detected using quantitative real-time PCR assay with a SYBR Green RealMasterMix kit (Tiangen, Beijing, China). GAPDH was used as an endogenous ‘housekeeping’ gene to normalize RNA levels across samples. The primer sequences for genes of interest and controls were listed in supporting table.

Three days after TBI, anesthesia was initiated with 5% isoflurane, spontaneous ventilation was maintained with 1.5% isoflurane in oxygen-enriched air (20% oxygen/80% air), and the mice were sacrificed by cutting their necks. Total RNA was extracted from the mouse brain tissues using an RNA extraction kit (BioTeck, Beijing, China) according to the manufacturer’s instructions. Samples were first treated with 3 U/mg RNase R (Epicenter, Madison, WI, USA) for 15 minutes at 37°C. The mRNAs, lncRNAs, and circRNAs were then reverse transcribed into cDNA using an RT Master Mix for real-time polymerase chain reaction (qPCR) Kit (MCE, Monmouth Junction, NJ, USA) following the manufacturer’s instructions. Next, the expression levels of selected DEmRNAs, DElncRNAs, and DEcircRNAs were determined by qPCR using SYBR® Green qPCR Master Mix (MCE). GAPDH was used as an endogenous reference transcript for mRNAs/lncRNAs/circRNAs. The 2^–ΔΔCt method (Poorghobadi et al., 2023) was used when measuring relative gene expression. The sequences of the primers used for qRT-PCR are shown in Table 1.