CHRNA9 and MICU1 Govern the Potency of the Mitochondrial Permeability Transition Pore in Necroptotic Oral Cancer Progression

Our study included 30 tissue samples in total, which included 15 samples from OSCC patients, 10 from OSMF patients, and 5 normal control samples. To explore the involvement of genes associated with the changes observed in OSMF tissue biopsy samples, we performed RNA sequencing (RNA seq) analysis of moderately advanced (OSMF-III) and advanced (OSMF-IV) conditions. Additionally, we obtained tissue biopsies from patients diagnosed with OSCC occurring concomitantly with OSMF. For sequencing, QIAGEN Pure mRNA beads enriched the mRNA. The bound mRNA was isolated and enriched after bead binding. Subsequently, FastSelect reagent was added for enrichment before heat fragmentation. RNA processing used RNase H-Reverse Transcriptase with random primers for first-strand synthesis. Second-strand synthesis used 5′ phosphorylated primers for strand-specific ligation. Adapters were added to inserts in a strand-specific manner. Libraries were purified with QIAseq CleanStart PCR reagents to remove previous NGS libraries. Prepared libraries were compatible with Illumina NGS systems (NovaSeqTM 6000) with specified read lengths and index reads based on index types. For bulk transcriptome analysis, sequencing data were in FastQ format. Data pre-processing involved quality checks, adapter removal, trimming, and data filtering. QC-passed samples were processed and analysed using the nf-core pipeline to generate a gene expression matrix. Differential expression gene (DEG) analysis was carried out with the iGeak RNA-seq software to normalize the raw gene expression data and analyse expression variances between the groups.