miRNA and mRNA Signatures in Human Acute Kidney Injury Tissue [mRNA, minimal change disease]
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 10 ng of total RNA were used for each sample's library preparation. Ribosomal RNA was removed using RiboGone – Mammalian Kit protocol. After rRNA depletion, double-stranded cDNA was synthesized and amplified using the SMARTer Universal Low Input RNA Kit protocol. The cDNA was sheared by Covaris, and the library was prepared and barcoded following the Ion Plus Fragment Library Kit protocol. Each resulting barcoded library was quantified and quality assessed by Agilent Bioanalyzer. Multiple libraries were pooled in equal molarity. Finally, 8 µL of 100 pM pooled libraries were loaded into lanes of an Illumina HiSeq 4000. At least 30M reads per library were generated. Overall design: Bulk 10 um thickness specimens from cross-sectional human kidney biopsies embedded in OCT underwent RNA sequencing.