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

Marc K Halushka,Karen Fox-Talbot,Carrie Wright,Joo Heon Shin,Courtney Williams,Anandita Rajpurohit,Avi Z Rosenberg,Olga Kovbasnjuk,Matthew N Mccall,Corey Porter

doi:10.1038/s41598-018-28198-z

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 epithelial-enriched 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 flow-isolated 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.

Highlights

MicroRNAs are small, regulatory RNA elements with critical control of protein expression
We introduce xMD-miRNA-seq, a method to obtain nearly in vivo miRNA expression estimates from any cell type directly from formalin-fixed paraffin-embedded (FFPE) tissues by utilizing expression microdissection[20]
Total RNA extraction and purification was performed on the cells annealed to the ethylene vinyl acetate (EVA) membranes yielding 266 (13.3 ng/μl) and 374 (18.7 ng/μl) ng of total RNA respectively for the two samples

Summary

Introduction

MicroRNAs (miRNAs) are small, regulatory RNA elements with critical control of protein expression. Several proliferation-related miRNAs of the miR-17-92 cluster were upregulated 3–6 fold over the same time course[10] These cell culture-mediated aberrations in relative miRNA expression levels can greatly impact disease-related studies. It is difficult to reconcile this difference other than to acknowledge that this miRNA, associated with a mature cell phenotype, is greatly reduced in a cell-culture sample[15]. To overcome this problem, there is a need for methods to capture in vivo cell expression miRNA estimates in a robust and cost-effective manner. Single-cell sequencing has great promise, current methodologies are limited for miRNAs due to cost, and depth of sequencing per cell[18]

Methods

Results

Conclusion