Expression Microdissection for the Analysis of miRNA in a Single-Cell Type

被引:1
|
作者
Jenike, Ana E. [1 ]
Bunkelman, Brady [1 ]
Mandell, Kira A. Perzel [2 ]
Oduor, Cliff I. [3 ]
Chin, Deborah [3 ]
Mair, Devin [4 ]
Jenike, Katharine M. [2 ]
Kim, Deok-Ho [4 ,5 ]
Bailey, Jeffrey A. [3 ]
Rafailovich, Miriam H. [6 ]
Rosenberg, Avi Z. [1 ]
Halushka, Marc K. [1 ]
机构
[1] Johns Hopkins Univ, Dept Pathol, Sch Med, Baltimore, MD 21205 USA
[2] Johns Hopkins Univ, Sch Med, Dept Genet Med, Baltimore, MD USA
[3] Brown Univ, Warren Alpert Med Sch, Dept Pathol & Lab Med, Providence, RI 02912 USA
[4] Johns Hopkins Univ, Dept Biomed Engn, Baltimore, MD USA
[5] Johns Hopkins Univ, Sch Med, Dept Med, Baltimore, MD USA
[6] SUNY Stony Brook, Dept Mat Sci & Chem Engn, Stony Brook, NY 11974 USA
关键词
tissue dissection; microRNA; methods; MIR-143/145; MICRORNAS; ATLAS;
D O I
10.1016/j.labinv.2023.100133
中图分类号
R-3 [医学研究方法]; R3 [基础医学];
学科分类号
1001 ;
摘要
Cell-specific microRNA (miRNA) expression estimates are important in characterizing the localization of miRNA signaling within tissues. Much of these data are obtained from cultured cells, a process known to significantly alter miRNA expression levels. Thus, our knowledge of in vivo cell miRNA expression estimates is poor. We previously demonstrated expression microdissectionmiRNA-sequencing (xMD-miRNA-seq) to acquire in vivo estimates, directly from formalin-fixed tissues, albeit with a limited yield. In this study, we optimized each step of the xMD process, including tissue retrieval, tissue transfer, film preparation, and RNA isolation, to increase RNA yields and ultimately show strong enrichment for in vivo miRNA expression by qPCR array. These method improvements, such as the development of a noncrosslinked ethylene vinyl acetate membrane, resulted in a 23- to 45-fold increase in miRNA yield, depending on the cell type. By qPCR, miR-200a increased by 14-fold in xMD-derived small intestine epithelial cells, with a concurrent 336-fold reduction in miR-143 relative to the matched nondissected duodenal tissue. xMD is now an optimized method to obtain robust in vivo miRNA expression estimates from cells. xMD will allow formalin-fixed tissues from surgical pathology archives to make theragnostic biomarker discoveries.& COPY; 2023 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.
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页数:9
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