miRNA and mRNA Signatures in Human Acute Kidney Injury Tissue [miRNA]
Acute kidney injury (AKI) is an important contributor to the development of chronic kidney disease (CKD). We performed miRNA and mRNA sequencing on biobanked human kidney tissues obtained in the routine clinical care of patients with the diagnoses of AKI and minimal change disease (MCD), in addition to nephrectomized (Ref) tissue from individuals without known kidney disease. From all renal biopsy samples, 2 cryosections (10 µM) including the entire cross-section of the tissue were placed directly into PicoPure RNA extraction buffer. RNA was isolated using the PicoPure isolation kit. Total RNA was evaluated for quantity and quality using an Agilent Bioanalyzer. Approximately 100 ng of total RNA was used for library construction with QIASeq miRNA Library Kit. Each resulting indexed library was quantified and its quality assessed by Qubit and Agilent Bioanalyzer. The libraries were then loaded onto an Illumina NextSeq 500 for 75 bp single-read sequencing to a depth of 15-20M reads per library. Sequence reads were uploaded to the Qiagen GeneGlobe Data Analysis Center (https://www.qiagen.com/us/resources/geneglobe) for quality control, alignment and expression quantification. Reads were mapped to different databases for mature, hairpin, noncoding RNA, mRNA and other RNA using bowtie v1.2. Read counts and UMI counts for each RNA category (mature, hairpin, piRNA, tRNA, rRNA, mRNA and otherRNA) were calculated for each sample, using miRbase V21 for miRNA, and piRNABank for piRNA. Overall design: Bulk 10 um thickness specimens from cross-sectional human kidney biopsies or nephrectomies embedded in OCT underwent small RNA sequencing. Biopsy subjects had AKI or minimal change disease, nephrectomy subjects had no known kidney disease.