A Transcriptome-wise Atlas of Human Prostate as a Function of Postmortem Interval Time
The prostate gland is one of the last internal organs to deteriorate during human decomposition. However, the effect of postmortem interval (PMI) on the mRNA and lncRNA expression and splicing is yet to be investigated in detail. The current study aims to identify the functional role of postmortem gene induction and pathway activation in prostate tissues with respect to the PMI gradient. The cadaver samples that were used in this study were collected during forensic autopsies and preserved at -20°C within the morgue at the University of Pavia (Pavia, Italy).  After RNA extraction, total RNA sequencing was performed on Illumina's NovaSeq 6000 using paired-ended sequencing approach. StringTie was used to perform expression level for mRNAs and lncRNAs by calculating FPKM. Additionally, mRNAs/lncRNAs differential expression analysis was performed by DESeq2. rMATS (version 4.1.1) was used to identify alternative splicing events and analyze differential alternative splicing events between samples having high and low PMI. Pathway analysis of the differentially expressed genes demonstrated the enrichment of FoxO signaling, aldosterone-regulated sodium reabsorption and adipocytokine signaling pathways in prostate tissue with high PMI. Gene Set Enrichment Analysis (GSEA) indicated the positive enrichment of genes belonging from protein export, proteasome, ferroptosis, and citric acid cycle in high PMI group. A comprehensive detection of alternative splicing events (ASEs) at the cellular level in postmortem prostate tissue was performed to reveal skipped exon events to be most prominent ASE in high PMI group followed by retained intron events.   Our study implies that the transcription machinery remains active in prostate tissue even after five days postmortem. Overall design: Prostate Tissue samples were removed from the prostate of five males with different causes of death. RNA was extracted using QIAGEN RNeasy Mini Kit (cat. no. 74104, Qiagen) from the prostate tissues (~ 30 mg). RNA extracts were quantitated using the Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA).For Total RNA sequencing service, RNA integrity was checked with Agilent Technologies 2100 Bioanalyzer. Ribosomal RNA was removed, followed by fragmentation with divalent cation buffers in elevated temperature. The sequencing library was prepared following Illumina's TruSeq-stranded-total-RNA-sample preparation protocol. Quality control analysis and quantification of the sequencing library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip. Paired-ended sequencing was performed on Illumina's NovaSeq 6000 sequencing system.