Non-coding RNA profiling by high throughput sequencing
Summary
Purpose: To identify the differentially expressed miRNA in lesional skin of psoriasis patients compared to adjacent normal tissue by Next Generation Sequencing. Methods: Small RNA isolated from 24 paired lesional and adjacent normal tissue sample and Next Generation Sequencing was performed. The sequences reads were trimmed of adapter sequences and quality trimmed using CutAdapt, followed by alignment to the human genome via 'Novoalign'. The aligned files were sorted based on chromosomal location and HTSeq-count was used to obtain the raw read counts for all known miRNAs. Differnetial expression analysis was performed with the raw reads using R-package 'edgeR'. MiRNA expression was quantified in tissue and serum by qRT-PCR method using SYBR-Green assay. Correlation analysis was done between miRNA expression in individual samples with their respective PASI scores to identify the miRNA associated with disease severity. Results: We identified 75 differentially expressed miRNA with at least 2-fold deregulation that were statistically significant. qRT-PCR validation showed approx 90% concordance with NGS data. Fold change of 4 miRNAs - miR-30b-5p, miR-7-1-3p, miR-33a-3p and miR-4742-3p were found to be asssociated with disease severity in psoriasis skin. Another miRNA let-7d-5p expression was found to be associated with disease severity in serum of HLA-Cw6 positive patients. Conclusions: Our study identified several unqiue deregulated miRNAs in psoriatric skin, which had not been previously reported. We also identified miRNA whose deregulated expression in psoriasis is associated with disease severity and the biological pathways through which they regulate disease pathogenesis.
Overall design
Small RNA profiling in 24 paired lesional psoriatic skin and adjacent normal skin
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