A simple high-throughput technology enables gain-of-function screening of human microRNAs

Wen Chih Cheng, Tami J. Kingsbury, Sarah J. Wheelan, Curt I. Civin

Research output: Contribution to journalArticlepeer-review

Abstract

MicroRNAs (miRs) regulate cellular processes by modulating gene expression. Although transcriptomic studies have identified numerous miRs differentially expressed in diseased versus normal cells, expression analysis alone cannot distinguish miRs driving a disease phenotype from those merely associated with the disease. To address this limitation, we developed mir-HTS, a method for unbiased high-throughput screening of the miRNome to identify functionally relevant miRs. Herein, we applied miR-HTS to simultaneously analyze the effects of 578 lentivirally transduced human miRs or miR clusters on growth of the IMR90 human lung fbroblast cell line. Growth-regulatory miRs were identified by quantitating the representation (i.e., relative abundance) of cells over expressing each miR over a one-month culture of IMR90, using a panel of custom-designed quantitative real-time PCR (qPCR) assays specific for each transduced miR expression cassette. Te miR-HTS identified 4 miRs previously reported to inhibit the growth of human lung-derived cell lines and 55 novel growth-inhibitory miR candidates. Nine of 12 (75%) selected candidate miRs were validated and shown to inhibit IMR90 cell growth. Thus, this novel lentiviral library- and qPCR-based miR-HTS technology provides a sensitive platform for functional screening that is straightforward and relatively inexpensive.

Original languageEnglish (US)
Pages (from-to)77-86
Number of pages10
JournalBioTechniques
Volume54
Issue number2
DOIs
StatePublished - Feb 2013

Keywords

  • Functional genomics
  • High-throughput screen
  • MicroRNA
  • Tumor suppressors
  • qPCR

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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