RNA Isolation from Mammalian Samples

Ximeng Liu1, Shuko Harada1

1 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.16
DOI:  10.1002/0471142727.mb0416s103
Online Posting Date:  July, 2013
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Abstract

RNA can be extracted from cultured cells, peripheral blood, bone marrow, plasma, serum, body fluids, and fresh or frozen tissues. RNA can also be extracted from formalin‐fixed paraffin‐embedded (FFPE) tissues. Methods for RNA extraction can be divided into three groups: phenol/chloroform extraction, silica spin‐column absorption, and isopycnic gradient centrifugation. Two different RNA isolation procedures are described in this unit. The first basic protocol describes a one‐step isolation involving monophasic lysis with guanidine isothiocyanate and phenol followed by chloroform extraction. The second basic protocol describes a silica‐column separation method for RNA isolation. Curr. Protoc. Mol. Biol. 103:4.16.1–4.16.16. © 2013 by John Wiley & Sons, Inc.

Keywords: RNA isolation; monophasic lysis reagent; spin column

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of RNA with Monophasic Lysis Reagent
  • Basic Protocol 2: Isolation of RNA with QIAamp RNA Blood Mini Kit
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of RNA with Monophasic Lysis Reagent

  Materials
  • Sample for RNA isolation (one of more of the following)
    • Plasma, serum, or whole blood, collected in EDTA [lavender‐top tube; store at 2° to 8°C for no more than 16 to 18 hr before extraction unless mixed with RNAlater (Qiagen)]
    • Cultured cells (monolayer or suspension)
    • Fresh or frozen tissue
    • Formalin‐fixed paraffin‐embedded (FFPE) samples
  • Monophasic Lysis Reagent (MLR; see Table 4.16.1)
  • Phosphate‐buffered saline (PBS; appendix 22; for cultured cells)
  • ATL buffer (Qiagen; for FFPE samples)
  • Proteinase K (Qiagen; for FFPE samples)
  • Chloroform
  • Isopropanol
  • 75% and 100% ethanol
  • RNase‐free water (DEPC‐treated water; unit 4.1)
  • Recombinant DNase I (rDNase I, 2 U/ l) and 10× buffer (Ambion)
  • 25:24:1 phenol/chloroform/isoamyl alcohol
  • RNase‐free disposable 1.5‐ml microcentrifuge tubes
  • (Optional) Cell scraper (for use with cells grown in monolayer)
  • Micropestle or homogenizer (for fresh or frozen tissue)
  • Additional reagents and equipment for isolation from FFPE tissue (unit 2.14) and RNA quantitation ( appendix 3D)

Basic Protocol 2: Isolation of RNA with QIAamp RNA Blood Mini Kit

  Materials
  • Blood or bone marrow collected in EDTA [lavender top tube; store at 2° to 8°C for no more than 16 to 18 hr before extraction unless mixed in RNAlater solution (Qiagen)]
  • Reagents from QIAamp RNA Blood Mini Kit (Qiagen)
    • Buffer EL
    • Buffer RLT (add 10 µl of 2‐ME to 1 ml prior to use)
    • Buffer RW1
    • Buffer RPE (add 44 ml of 96% to 100% ethanol to the concentrate prior to use)
    • QIAshredder spin column
    • 2‐ml collection tubes
    • QIAamp spin column
  • 2‐mercaptoethanol (2‐ME)
  • 70% ethanol
  • RNase‐free water (DEPC‐treated water; unit 4.1)
  • 15‐ml conical tubes
  • RNase‐free pipet
  • Aerosol‐resistant pipet tips
  • 1.5‐ml microcentrifuge tubes (RNase‐free)
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Figures

Videos

Literature Cited

   Chirgwin, J.M., Przybyla, 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.
   Chomczynski, P. 1993. A reagent for the single‐step isolation of viral RNA from human serum and biopsy samples. Biotechniques 15:532‐537.
   Chomczynski, P. and Sacchi, N. 1987. Single‐step method of RNA isolation by acid guanidinium thiocyanate‐phenol‐chloroform extraction. Anal. Biochem. 162:156‐159.
   Kirby, K.S. 1968. Isolation of nucleic acids with phenolic solvents. Methods Enzymol. 12B:87‐99.
   Kurland, C.G. 1960. Molecular characterization of ribonucleic acid from Escherichia coli ribosomes. I. Isolation and molecular weights. J. Mol. Biol. 2:83‐91.
Internet Resources
   http://www.invitrogen.com/site/us/en/home/Products‐and‐Services/Applications/DNA‐RNA‐Purification‐Analysis/rna‐extraction.html
   http://www.qiagen.com/default.aspx
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