RNA Extraction and Preparation in Rice (Oryza sativa)

Cui‐Cui Yin1, Biao Ma1, Wei Wang1, Qing Xiong1, He Zhao1, Shou‐Yi Chen1, Jin‐Song Zhang1

1 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
Publication Name:  Current Protocols in Plant Biology
Unit Number:   
DOI:  10.1002/cppb.20023
Online Posting Date:  June, 2016
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Abstract

There has been a surge of interest in the molecular biology of RNA. We describe a simple, fast, and convenient protocol for isolating total RNA from rice (Oryza sativa) seedlings and young tissues/organs. RNA obtained using this method can be used for reverse transcription‐polymerase chain reaction (RT‐PCR), quantitative RT‐PCR (qRT‐PCR), northern blot analysis, RNA‐seq, microarray analysis, cDNA library construction, etc. Technical parameters critical to the success of the extraction are presented. © 2016 by John Wiley & Sons, Inc.

Keywords: rice; RNA isolation; total RNA

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Extraction of Total RNA from Rice Seedlings and Young Tissues
  • Support Protocol 1: Preparation of RNase‐Free Labware and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Extraction of Total RNA from Rice Seedlings and Young Tissues

  Materials
  • Rice seeds or young tissue/organ (freshly harvested or frozen at −80°C)
  • RNase‐free water
  • Hoagland solution (PhytoTechnology Laboratories, cat. no. 15L0353029A) or nutrient soil
  • Liquid nitrogen
  • TRIzol reagent (Invitrogen, cat. no. 15596‐026)
  • Chloroform
  • Isopropyl alcohol
  • 75% RNase‐free ethanol
  • Ice
  • 200‐ml beakers or 250‐ml flasks, according to the amount of seeds needed
  • 37°C incubator
  • 96‐well plates specially made for growing rice seedlings, with bottom‐less wells and fitted with 96‐well floater
  • Mortar and pestle or electric drill
  • Medicine spoon
  • RNase‐free pipet tips and microcentrifuge tubes (Axygen)
  • Centrifuge (Thermo Scientific LEGEND MICRO 17 R)
  • Water bath or heat block
  • Spectrophotometer (NanoDrop1000, Thermo Scientific or Eppendorf Biophotometer plus)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Support Protocol 1: Preparation of RNase‐Free Labware and Solutions

  Materials
  • Diethyl pyrocarbonate (DEPC)
  • Mortar
  • Pestle
  • Beaker
  • Pipet tips
  • 1.5‐ml microcentrifuge tube
  • Aluminum foil
  • Oven
  • Autoclave
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Figures

Videos

Literature Cited

Literature Cited
  Che, R.H., Tong, H.N., Shi, B.H., Liu, Y.Q., Fang, S.R., Liu, D.P., Xiao, Y.H., Hu, B., Liu, L.C., Wang, H.R., Zhao, M.F., and Chu, C.C. 2015 Control of grain size and rice yield by GL2‐mediated brassinosteroid responses. Nat. Plants doi: 10.1038/nplants.2015.195.
  Chirgwin, J.M., Pryzbyla, A.E., MacDonald, R.J., and Rutter, W.J. 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294‐5299. doi: 10.1021/bi00591a005.
  Chomczynski, P. and Sacchi, N. 1987 Single step method of RNA isolation by acid guanidinium thiocyanate‐phenol‐chloroform extraction. Anal. Biochem. 163:156‐159. doi: 10.1016/0003‐2697(87)90021‐2.
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  Ma, B., He, S.J., Duan, K.X., Yin, C.C., Chen, H., Yang, C., Xiong, Q., Song, Q.X., Lu, X., Chen, H.W., Zhang, W.K., Lu, T.G., Chen, S.Y., and Zhang, J.S. 2013. Identification of rice ethylene‐response mutants and characterization of MHZ7/OsEIN2 in distinct ethylene response and yield trait regulation. Mol. Plant 6:1830‐1848. doi: 10.1093/mp/sst087.
  Suzuki, Y., Makino, A., and Mae, T. 2001 An efficient method for extraction of RNA from rice leaves at different ages using benzyl chloride. J. Exp. Bot. 52:1575‐1579. doi: 10.1093/jexbot/52.360.1575.
  Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
  Wang, S.K., Li, S., Liu, Q., Wu, K., Zhang, J.Q, Wang, S.S., Wang, Yi., Chen, X.B., Zhang, Y., Gao, C.X., Wang, F., Huang, H.X., and Fu, X.D. 2015. The OsSPL16‐GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality. Nat. Genet. 47:949‐954. doi: 10.1038/ng.3352.
  Yang, C., Ma, B., He, S.J., Xiong, Q., Duan, K.X., Yin, C.C., Chen, H., Lu, X., Chen, S.Y., and Zhang, J.S. 2015. MAOHUZI6/ETHYLENE INSENSITIVE3‐LIKE1 and ETHYLENE INSENSITIVE3‐LIKE2 regulate ethylene response of roots and coleoptiles and negatively affect salt tolerance in rice. Plant Physiol. 169:148‐165. doi: 10.1104/pp.15.00353.
  Yin, C.C., Ma, B., Collinge, D., Pogson, B., He, S.J., Xiong, Q., Duan, K.X., Chen, H., Yang, C., Lu, X., Wang, Y.Q., Zhang, W.K., Chu, C.C., Sun, X.H., Fang, S., Chu, J.F., Lu, T.G., Chen, S.Y., and Zhang, J.S. 2015 Ethylene responses in rice roots and coleoptiles are differentially regulated by a carotenoid isomerase‐mediated Abscisic Acid pathway. Plant Cell 27:1061‐1081. doi: 10.1105/tpc.15.00080.
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