Capture and Identification of miRNA Targets by Biotin Pulldown and RNA-seq

Shen Mynn Tan; Judy Lieberman

doi:10.1007/978-1-4939-3067-8_13

Capture and Identification of miRNA Targets by Biotin Pulldown and RNA-seq

Methods Mol Biol. 2016:1358:211-28. doi: 10.1007/978-1-4939-3067-8_13.

Authors

Shen Mynn Tan^{1

2}, Judy Lieberman^{3

4}

Affiliations

¹ Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA, 02115, USA.
² Department of Pediatrics, Harvard Medical School, 200 Longwood Ave, Boston, MA, 02115, USA.
³ Cellular and Molecular Medicine Program, Boston Children's Hospital, Boston, MA, 02115, USA. Judy.Lieberman@childrens.harvard.edu.
⁴ Department of Pediatrics, Harvard Medical School, 200 Longwood Ave, Boston, MA, 02115, USA. Judy.Lieberman@childrens.harvard.edu.

PMID: 26463386
DOI: 10.1007/978-1-4939-3067-8_13

Abstract

MicroRNAs (miRNAs) are small noncoding RNAs that regulate the stability and expression of target RNAs in a sequence-dependent manner. Identifying miRNA-regulated genes is key to understanding miRNA function. Here, we describe an unbiased biochemical pulldown method to identify with high-specificity miRNA targets. Regulated transcripts are enriched in streptavidin-captured mRNAs that bind to a transfected biotinylated miRNA mimic. The method is relatively simple, does not involve cross-linking and can be performed with only a million cells. Addition of an on-bead RNase digestion step also identifies miRNA recognition elements (MRE).

Keywords: MicroRNA; Noncoding RNA; Target identification; miRNA recognition element.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Biotin / chemistry
High-Throughput Nucleotide Sequencing / methods*
MicroRNAs / genetics*
MicroRNAs / isolation & purification
RNA, Messenger / genetics*
RNA, Messenger / isolation & purification
Ribonucleases / genetics
Streptavidin / chemistry

Substances

MicroRNAs
RNA, Messenger
Biotin
Streptavidin
Ribonucleases