xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

Abstract

Accurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelialenriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flowisolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

Consistent RNA sequencing contamination in GTEx and other data sets

“…Another challenge of high-throughput RNA-Seq is contamination, leading to the presence of sequence data within a data set of one sample that originates from a separate sample. This contamination can come from many different aspects of the modern sequencing process, such as human error, machine or equipment contamination, intrinsic preparation and sequencing errors, and computational errors, including errors that can occur based on the multiplexing methods used (https://www.illumina.com/content/dam/illumina-marketing/ documents/products/whitepapers/index-hopping-white-paper-770-2017-004.pdf, visited 02/24/2020) 10,11 . For single cell RNA-Seq (scRNA-Seq), doublets or multiplets (2+ cells partitioned together) can cause cross-contamination resulting in expression hybrids 12 .…”