Multiprobe Ribonuclease Protection Assay for Simultaneous Measurement of mRNA Expression

Howard A. Young1, Jeffrey J. Subleski1, Stephanie M. Krebs1

1 National Cancer Institute, Frederick, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 10.29
DOI:  10.1002/0471142735.im1029s54
Online Posting Date:  August, 2003
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The multiprobe RNase protection assay enables investigators to monitor RNA expression of 8‐12 genes with as little as 1 µg of total RNA. The commercial availability of numerous multi‐gene template sets makes this assay practical for all basic research programs.

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Table of Contents

  • Basic Protocol 1: Multiprobe Ribonuclease Protection Assay
  • Support Protocol 1: Constructing a Riboprobe Template Set
  • Support Protocol 2: Gel Electrophoresis and Visualization of Results
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Multiprobe Ribonuclease Protection Assay

  • RiboQuant In Vitro Transcription Kit (BD PharMingen):
  •  40 U/µl RNasin
  •  GACU pool (2.75 mM GTP/2.75 mM ATP/2.75 mM CTP/61 µM UTP)
  •  100 mM DTT
  •  5× transcription buffer
  •  20 U/µl T7 RNA polymerase
  •  1 U/µl RNase‐free DNase
  •  2 mg/ml yeast tRNA
  •  20 mM EDTA
  •  4 M ammonium acetate
  • DEPC‐treated water (unit 12.8)
  • 5 to 10 ng/µl RiboQuant Multiprobe template set (BD PharMingen) and/or custom set (see protocol 2)
  • 3000 Ci/mmol [33P]UTP(New England Nuclear) or [32P]UTP
  • Amersham MicroSpin G25 columns
  • Scintillation fluid (for 33P‐labeled probes)
  • Total RNA (unit 10.11)
  • RPA kit (BD PharMingen)
  •  1× hybridization buffer (BD PharMingen)
  •  2 mg/ml yeast tRNA
  •  1× RNase buffer (BD PharMingen)
  •  RNase A + T1 mix (80 ng/µl RNase A/250 U/µl RNase T1)
  •  1× proteinase K buffer
  •  10 mg/ml proteinase K
  •  4 M ammonium acetate
  • Mineral oil
  • RNase Inactivation/PPT Reagent III (Ambion)
  • 100% ethanol
  • GlycoBlue coprecipitant (Ambion)
  • 0.5‐ and 1.5‐ml microcentrifuge tubes, sterile
  • 1.5‐ and 2.2‐ml microcentrifuge tubes
  • Scintillation vials
  • Adjustable heating block (e.g., Boekel model 111007)
  • Cotton swab, sterile
  • Additional reagents and equipment for gel electrophoresis and autoradiography (see protocol 3)
CAUTION: Radioactive materials require special handling. See appendix 1Q and the institutional Radiation Safety Office for guidelines concerning proper handling and disposal.

Support Protocol 1: Constructing a Riboprobe Template Set

  • Target cDNA
  • SuperScript One‐Step RT‐PCR kit (Invitrogen; optional)
  • Primers:
  •  Sense primer for sequence of interest (e.g., Invitrogen, Operon)
  •  T7 polymerase primer (e.g., Invitrogen, Operon)
  • TA or Zero Blunt TOPO Cloning kit with PCR 4 cloning vector (Invitrogen; optional)
  • Appropriate host strain
  • LB plate (unit 10.19) with appropriate selectivity
  • Invitrogen Platinum PCR Supermix
  • DNA ladder (e.g., DNA low‐mass ladder, Invitrogen)
  • Spin column (e.g., GenElute Minus EtBr spin column, Sigma)
  • 5 M ammonium acetate, pH 5.3 or 3 M sodium acetate, pH 5.5
  • 100% ethanol
  • 70% ethanol, 4°C
  • Ambion T7 RNA polymerase/RNase Inhibitor Maxiscript
  • Pipet tips, sterile
  • 94°C water bath or heating block
  • Additional reagents and equipment for RT‐PCR (unit 10.23), transforming bacteria ( appendix 3N), PCR (unit 10.20), agarose gel electrophoresis (unit 10.4), isolating and purifying DNA from an agarose gel (unit 10.5), and restriction digestion (unit 10.8)
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Literature Cited

Literature Cited
   Hallensleben, W., Biro, L., Sauder, C., Hausmann, J., Asensio, V.C., Campbell, I.L., and Staeheli, P. A polymorphism in the mouse crg‐2/IP‐10 gene complicates chemokine gene expression analysis using a commercial ribonuclease protection assay. 2000. J. Immunol. Methods. 234:149‐151.
   Harju, S. and Peterson, K.R. Sensitive ribonuclease protection assay employing glycogen as a carrier and a single inactivation/precipitation step. 2001. Biotechniques 30:1198‐1204.
   Kono, D.H., Baccala, R., Balderas, R.S., Kovac, S.J., Heald, P.W., Edelson, R.L., and Theofilopoulos, A.N. Application of a multiprobe RNase protection assay and junctional sequences to define V beta gene diversity in Sezary syndrome. 1992. Am. J. Pathol. 140:823‐830.
   Krupp, G., RNA synthesis: Strategies for the use of bacteriophage RNA polymerases. 1988. Gene 72:75‐89.
   Luckow, B., Maier, H., Chilla, S., and Perez de Lema, G., The mCK‐5 multiprobe RNase protection assay kit can yield erroneous results for the murine chemokines IP‐10 and MCP‐1. 2000. Anal. Biochem. 286:193‐197.
   Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., and Green, M.R. 1984. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucl. Acids Res. 12:7035‐7056.
   Muller, K., Ehlers, S., Solbach, W., and Laskay, T. Novel multi‐probe RNase protection assay (RPA) sets for the detection of murine chemokine gene expression. 2001. J. Immunol. Methods 249:155‐165.
   Okada, C.Y. and Weissman, I.L. 1989. Relative V beta transcript levels in thymus and peripheral lymphoid tissues from various mouse strains. Inverse correlation of I‐E and Mls expression with relative abundance of several V beta transcripts in peripheral lymphoid tissues. J. Exp. Med. May. 169:1703‐1719.
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