Analysis of Small Endogenous RNAs

Nelson C. Lau1

1 Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 26.7
DOI:  10.1002/0471142727.mb2607s81
Online Posting Date:  January, 2008
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Eukaryotic cells express small noncoding RNAs to silence target genes or regulate chromatin domains. MicroRNAs (miRNA) are endogenous small RNAs that are thought to each regulate multiple mRNA targets. To gain a deeper understanding of processes regulated by small noncoding RNAs, techniques are required to identify and detect them. This unit describes standard laboratory methods for and comments about high‐throughput technologies for the identification and detection of small RNAs. This unit also outlines a cell‐based reporter gene assay for assessing the regulatory potential of a microRNA on a candidate mRNA target. Curr. Protoc. Mol. Biol. 81:26.7.1‐26.7.15. © 2008 by John Wiley & Sons, Inc.

Keywords: Small RNA; RNAi; miRNA; microRNA regulation; gene silencing

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Table of Contents

  • Introduction
  • Basic Protocol 1: Simple Chromatography and Detection of Small‐RNA Ribonucleoproteins
  • Support Protocol 1: Increasing Small‐RNA Northern Blot Sensitivity by Chemical Cross‐Linking
  • Support Protocol 2: Assaying Regulatory Potential of Predicted miRNA Target Elements
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Simple Chromatography and Detection of Small‐RNA Ribonucleoproteins

  • Cells of interest, cultured or from tissue
  • 0.1 M and 1 M potassium acetate in base buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (unit 2.1)
  • Chloroform
  • 10 mg/ml glycogen from blue mussel
  • 3 M sodium chloride
  • Absolute and 70% (v/v) ethanol
  • Yeast poly‐A polymerase and 5× poly‐A polymerase buffer (USB)
  • 3′‐Deoxyadenosine 5′‐[α‐32P]‐triphosphate (cordycepin 5′‐[α‐32P]‐triphosphate, 5000 Ci/mmol, 185 TBq/mmol; Perkin Elmer)
  • Formamide loading dye (unit 4.9)
  • 5‐ml Hitrap Q column (GE Healthcare)
  • FPLC instrument with fraction collector
  • 85°C heating block or water bath
  • Phosphorimager scanner or photographic film
  • Additional reagents and equipment for extracting soluble proteins from cells (unit 12.1), dialyzing extracts ( appendix 3C), extracting and precipitating nucleic acids (unit 2.1), and performing denaturing polyacrylamide gel electrophoresis with RNA (unit 4.9)

Support Protocol 1: Increasing Small‐RNA Northern Blot Sensitivity by Chemical Cross‐Linking

  • Small‐RNA samples (e.g., see protocol 1)
  • 12.5 M 1‐methylimidazole
  • RNase‐free water
  • 1 M HCl
  • 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC)
  • Neutral nylon membrane (e.g., Hybond NX, GE Healthcare)
  • 3MM blotting paper, slightly larger than membrane (20 × 16–cm maximum)
  • Glass plate
  • Plastic wrap (e.g., Saran Wrap)
  • 50°C to 60°C incubator
  • Additional reagents and equipment for performing denaturing polyacrylamide gel electrophoresis and northern blots (unit 4.9)

Support Protocol 2: Assaying Regulatory Potential of Predicted miRNA Target Elements

  • Synthetic miRNA duplexes: experimental and noncognate control (completely different sequence)
  • Genomic DNA or cDNA containing wildtype (wt) and mutant UTR elements (see unit 8.5)
  • TK– or SV40–Renilla luciferase reporter gene plasmid (Promega)
  • Mammalian cells (e.g., HeLa cells)
  • Lipofectamine 2000 (Invitrogen)
  • SV40–firefly luciferase control plasmid (Promega)
  • pUC19 (carrier DNA plasmid)
  • PBS ( appendix 22)
  • Commercial cell lysis buffer (e.g., Promega)
  • Dual‐luciferase luminescence detection kit (Promega)
  • 24‐well culture plate
  • Luminometer
  • Additional reagents and equipment for PCR (unit 15.1), subcloning (unit 3.16), sequencing (Chapter 7), purifying by anion‐exchange chromatography (unit 2.1), growing mammalian cells in culture ( appendix 3F), and cotransfecting siRNA and plasmids (unit 26.2)
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Literature Cited

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