Small RNA Extraction and Detection in Rice (Oryza sativa)

Xianwei Song1, Xiaofeng Cao1

1 State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20005
Online Posting Date:  May, 2016
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Rice (Orzya sativa) small RNAs regulate almost all biological processes and agronomic traits. Characterization and dissection of small RNA functions have become an important part of rice functional genomic research. Isolation of small RNAs of desirable quality is essential for a variety of analytical techniques such as cloning, RT‐PCR, northern hybridization, and microarray analysis. Among these, northern hybridization is the most convincing method to display size distribution, abundance, and expression profiles of small RNAs. This article describes protocols for extraction and detection of small RNAs from rice tissues, including procedures to enrich small RNAs from total RNA, and examination of the size and abundance of small RNAs by northern hybridization. © 2016 by John Wiley & Sons, Inc.

Keywords: northern hybridization; rice; Orzya sativa; small RNA

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Table of Contents

  • Introduction
  • Basic Protocol 1: Small RNA Extraction and Enrichment
  • Basic Protocol 2: Detection of Small RNAs by Northern Hybridization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Small RNA Extraction and Enrichment

  • Rice tissue
  • Liquid nitrogen
  • Diethylpyrocarbonate (DEPC)‐treated water (see recipe)
  • TRIzol Reagent (Invitrogen)
  • Chloroform
  • 75% and 100% ethanol (prepared with DEPC‐treated water)
  • 3 M sodium acetate solution (see recipe)
  • Water‐saturated phenol (optional; for samples with high polysaccharide, protein, or lipid content)
  • 8 M LiCl solution (see recipe)
  • Deionized formamide
  • Mortar and pestle
  • 1.5‐ and 2.0‐ml microcentrifuge tubes
  • Centrifuge
  • −20°C freezer
  • Vortex
  • UV‐vis spectrophotometer (Thermo Scientific, NanoDrop 1000)

Basic Protocol 2: Detection of Small RNAs by Northern Hybridization

  • Enriched small RNA samples (see protocol 1)
  • 15% (w/v) polyacrylamide/7 M urea solution (see recipe)
  • 10% (w/v) ammonium persulfate
  • RNA loading buffer (see recipe)
  • 0.25× and 0.5× TBE (see recipe)
  • Ethidium bromide
  • Neutral nylon membrane (GE Healthcare, Hybond‐NX)
  • EDC cross‐linking solution, prepare fresh (see recipe)
  • 10 μM DNA oligonucleotide
  • 10× T4 kinase buffer
  • DEPC‐treated water
  • γ‐32P ATP
  • 10 U/μl T4 polynucleotide kinase
  • 10× transcription buffer
  • 40 U/μl RNasin (e.g., Promega)
  • 10 mM each rATP, rGTP, and rCTP
  • RNA template
  • α‐32P UTP
  • T7 RNA polymerase
  • 10 U/μl DNase I (RNase‐free)
  • 120 mM sodium carbonate/80 mM sodium bicarbonate solution
  • 3 M sodium acetate solution (see recipe)
  • Hybridization solution (see recipe)
  • 2× SSC/0.2% (w/v) SDS solution
  • Gel casting platform
  • Heating block
  • Equipment for gel electrophoresis
  • Gel imaging platform (e.g., BioRad, GelDoc XR+)
  • Semi‐dry transfer unit (e.g. GE Healthcare, Hoefer TE77)
  • Whatman 3M filter paper
  • Plastic wrap
  • Storage phosphor screen
  • Imaging system for radioactive samples (e.g., GE Healthcare, Typhoon Trio)
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Literature Cited

Literature Cited
  Pall, G.S. and Hamilton, A.J. 2008. Improved northern blot method for enhanced detection of small RNA. Nat. Protoc. 3:1077‐1084. doi: 10.1038/nprot.2008.67.
  Pall, G.S., Codony‐Servat, C., Byrne, J., Ritchie, L., and Hamilton, A. 2007. Carbodiimide‐mediated cross‐linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res. 35:e60. doi: 10.1093/nar/gkm112.
  Sunkar, R., Girke, T., Jain, P.K., and Zhu, J.K. 2005a. Cloning and characterization of microRNAs from rice. Plant Cell 17:1397‐1411. doi: 10.1105/tpc.105.031682.
  Sunkar, R., Girke, T., and Zhu, J.K. 2005b. Identification and characterization of endogenous small interfering RNAs from rice. Nucleic Acids Res. 33:4443‐4454. doi: 10.1093/nar/gki758.
  Vester, B. and Wengel, J. 2004. LNA (locked nucleic acid): High‐affinity targeting of complementary RNA and DNA. Biochemistry 43:13233‐13241. doi: 10.1021/bi0485732.
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