RNA Analysis by Nuclease Protection

Marianna Goldrick1, Donald Kessler2

1 Ambion, Inc., Austin, Texas, 2 Genomics Corp., Foster City, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 5.1
DOI:  10.1002/0471142301.ns0501s23
Online Posting Date:  August, 2003
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Abstract

Nuclease protection assays (S1 nuclease protection and RNase protection) are extremely sensitive procedures for detection and quantitation of mRNA species in complex mixtures of total cellular RNA. These assays are well suited for mapping positions of external and internal junctions in RNA, such as transcription initiation and termination sites and intron/exon boundaries, and to discriminate between closely related targets by using probes designed to span the regions where the related genes differ the most. Also, because the size of the probes used in nuclease protection assays is a variable chosen by the investigator, probes may be designed to protect fragments of different sizes. This feature permits the simultaneous analysis of several different mRNAs in the same total RNA sample. In this unit, a method is included for RNase protection of target mRNA sequences, including hybridization of the probe to the target sequence, details of the actual protection assay, and detection of reaction products. An alternative method is provided for performing the RNase protection assay on a microvolume scale, which is useful when there are many samples to be analyzed. Support protocols describe synthesis and gel purification of labeled RNA probes; preparation of RNase‐free yeast RNA, which acts as an aid in the quantitative precipitation of newly synthesized probe; and quantitation of target mRNA. A method describing S1 nuclease protection of target mRNA using either RNA or DNA probes is also included. Additional support protocols provide instructions for the preparation of radiolabeled DNA probes by primer‐extension of double‐stranded plasmid or PCR product using Klenow fragment of E. coli DNA polymerase I or Taq or Tth polymerase in a thermal cycler. Another radiolabeling method details 5' end labeling of oligodeoxynucleotides and oligoribonucleotides using T4 polynucleotide kinase. Additionally, a method is described for mapping transcription start sites using the S1 nuclease protection assay.

     
 
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Table of Contents

  • Basic Protocol 1: RNase Protection Assay
  • Alternate Protocol 1: Small‐Volume RNase Protection Assay
  • Support Protocol 1: Synthesis and Gel Purification of Full‐Length RNA Probe
  • Support Protocol 2: Preparation of RNase‐Free Sheared Yeast RNA
  • Support Protocol 3: Absolute Quantitation of mRNA
  • Basic Protocol 2: Protection of mRNA from S1 Nuclease Digestion Using Single‐Stranded DNA or RNA Probes
  • Support Protocol 4: Synthesis of DNA Probes by Primer Extension of Double‐Stranded Plasmid or PCR Product Using Klenow Fragment
  • Support Protocol 5: Synthesis of DNA Probes by Primer Extension of Double‐Stranded Plasmid or PCR Product in a Thermal Cycler Using Thermostable Polymerase
  • Support Protocol 6: Synthesis of DNA Probes by T4 Polynucleotide Kinase End Labeling of Oligonucleotids
  • Support Protocol 7: 5′ End Mapping of mRNA Transcription Start Sites
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: RNase Protection Assay

  Materials
  • Radiolabeled RNA probe (see protocol 3) in probe elution buffer or hybridization buffer (see reciperecipes)
  • RNA sample: aqueous or in hybridization buffer (see recipe; storage in high concentrations of formamide enhances stability; Chomczynski, )
  • RNase‐free total yeast RNA (see protocol 4) or tRNA that does not contain the target sequence
  • 5 M ammonium acetate
  • 100% ethanol or isopropanol
  • Hybridization buffer (see recipe)
  • RNase A/RNase T1 stock solution (see recipe)
  • RNase digestion buffer (see recipe)
  • Proteinase K/SDS solution (see recipe)
  • 1 mg/ml carrier nucleic acid or glycogen
  • 25:24:1 phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • Gel loading buffer ( appendix 2A)
  • RNase‐free microcentrifuge tubes (e.g., Ambion)
  • Pasteur pipets with drawn‐out tips or fine‐gauge needles
  • Heating block
  • 42° to 45°C incubator or water bath
  • Denaturing 5% (19:1 acrylamide/bisacrylamide) polyacrylamide gel (CPMB UNIT )
  • Chromatography paper (Whatman)
  • Plastic wrap (if using 32P‐labeled probe)
  • X‐ray film (Kodak XRP)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (CPMB UNIT ) and autoradiography (CPMB APPENDIX )
CAUTION: Phenol and chloroform are severe health hazards. Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of appropriately.NOTE: Use DEPC‐treated water ( appendix 2A) to prepare all solutions.

Alternate Protocol 1: Small‐Volume RNase Protection Assay

  • RNA sample in volume ≤20 µl (preferably in hybridization buffer; see recipe)
  • Radiolabeled RNA probe (see protocol 3) in volume ≤20 µl (preferably in hybridization buffer; see recipe)

Support Protocol 1: Synthesis and Gel Purification of Full‐Length RNA Probe

  Materials
  • DEPC‐treated H 2O ( appendix 2A)
  • 10× transcription buffer (see recipe)
  • 3NTP mix (see recipe)
  • Dilute solution of limiting nucleotide: e.g., 50 µM cold UTP or CTP
  • 10 to 20 mCi/ml [α‐32P]UTP or [α‐32P]CTP (400 to 800 Ci/mmol) in aqueous buffer (not in ethanol)
  • 10 to 50 U/µl placental ribonuclease inhibitor (Ambion or Boehringer Mannheim)
  • Template DNA: 0.5 µg/ml linearized plasmid or 50 ng/ml amplified PCR product
  • 10 to 20 U/µl bacteriophage RNA polymerase (T7, T3, or SP6) appropriate to promoter used in template DNA (keep on ice before use)
  • 1 to 2 U/µl RNase‐free DNase I
  • Gel loading buffer ( appendix 2A)
  • 5% denaturing polyacrylamide gel (CPMB UNIT )
  • Probe elution buffer (see recipe)
  • 5 M ammonium acetate
  • Nucleic acid precipitation aid: 5 mg/ml RNase‐free yeast RNA (see protocol 4) or 5 mg/ml glycogen
  • 100% isopropanol (ACS grade)
  • Hybridization buffer (see recipe)
  • RNase‐free microcentrifuge tubes
  • Heating block
  • Scalpel
  • Forceps
  • Pasteur pipet with drawn‐out tip or fine‐gauge needle
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (CPMB UNIT ), autoradiography (CPMB APPENDIX ), and TCA precipitation (CPMB UNIT )
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of appropriately.NOTE: Use DEPC‐treated water ( appendix 2A) to prepare all solutions.

Support Protocol 2: Preparation of RNase‐Free Sheared Yeast RNA

  Materials
  • Total yeast RNA (from Torula yeast; Sigma)
  • Sodium dodecyl sulfate (SDS)
  • Proteinase K
  • 5 M ammonium acetate
  • 100% ethanol
  • 0.1 mM EDTA in DEPC‐treated H 2O ( appendix 2A)
  • 55°C water bath
  • Additional reagents and equipment for phenol/chloroform extraction and ethanol precipitation of RNA (see CPMB UNIT )
CAUTION: Phenol and chloroform are severe health hazards.NOTE: Use DEPC‐treated water ( appendix 2A) to prepare all solutions.

Support Protocol 3: Absolute Quantitation of mRNA

  Materials
  • Sample RNA
  • Labeled probe (see protocol 3, protocol 74, protocol 85, or protocol 96)
  • RNase‐free total yeast RNA (see protocol 4)
  • 5 M ammonium acetate
  • 100% ethanol, ice‐cold
  • Hybridization buffer (see recipe)
  • 2× S1 nuclease digestion buffer (see recipe)
  • DEPC‐treated H 2O ( appendix 2A)
  • 250 to 500 U/µl S1 nuclease (Boehringer Mannheim or Pharmacia Biotech)
  • S1 nuclease stop buffer (see recipe)
  • Gel loading buffer ( appendix 2A)
  • RNase‐free microcentrifuge tubes (e.g., Ambion)
  • Heating block
  • 42° to 45°C incubator or water bath
  • Pasteur pipets with drawn‐out tips
  • Chromatography paper (Whatman)
  • Plastic wrap
  • X‐ray film (Kodak XRP)
  • Additional reagents and equipment for denaturing polyacrylamide gel electrophoresis (see CPMB UNIT ) and autoradiography (see CPMB APPENDIX )
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of appropriately.NOTE: Use DEPC‐treated water ( appendix 2A) to prepare all solutions.

Basic Protocol 2: Protection of mRNA from S1 Nuclease Digestion Using Single‐Stranded DNA or RNA Probes

  Materials
  • Template DNA (linearized plasmid or purified PCR product)
  • Primer oligonucleotide (typically 20 bases in length)
  • DEPC‐treated H 2O ( appendix 2A)
  • Liquid nitrogen or dry ice/methanol bath
  • 10× primer‐extension buffer (see recipe)
  • 10 mCi/ml [α‐32P]dATP or [α‐32P]dCTP (3000 Ci/mmol)
  • 10× 3dNTP mix (appropriate to radiolabeled dNTP used; see recipe)
  • 50 mM dithiothreitol (DTT)
  • 5 U/µl Klenow fragment of E. coli DNA polymerase I
  • 25 mM recipeEDTA, pH 8.0 ( appendix 2A)
  • 25:24:1 phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • 5 M ammonium acetate
  • 100% ethanol, ice‐cold
  • Gel loading buffer ( appendix 2A)
  • 0.5‐ml RNase‐free microcentrifuge tubes
  • 95° to 100°C heating block or water bath
  • Additional reagents and equipment for gel purifying probes (see protocol 3)
CAUTION: Phenol and chloroform are severe health hazards. Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of appropriately.NOTE: Use DEPC‐treated water ( appendix 2A) to prepare all solutions.

Support Protocol 4: Synthesis of DNA Probes by Primer Extension of Double‐Stranded Plasmid or PCR Product Using Klenow Fragment

  Materials
  • Template DNA (linearized double‐stranded plasmid or PCR product)
  • 10× PCR buffer (see recipe)
  • 5 µM primer DNA (see CPMB UNIT , Critical Parameters, for guidelines on designing primers for PCR)
  • 10× 3dNTP mix (appropriate to labeled dNTP used; see recipe)
  • 10 mCi/ml [α‐32P]dATP or [α‐32P]dCTP (3000 Ci/mmol)
  • 2 U/µl Taq or Tth DNA polymerase
  • DEPC‐treated H 2O ( appendix 2A)
  • Mineral oil
  • 25 mM recipeEDTA, pH 8.0 ( appendix 2A)
  • 25:24:1 phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • 5 M ammonium acetate
  • 100% ethanol, ice‐cold
  • Gel loading buffer ( appendix 2A)
  • RNase‐free microcentrifuge tubes
  • Thermal cycler
  • Pasteur pipets with drawn‐out tips
  • Additional reagents and equipment for gel purifying probes (see protocol 3)
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of appropriately.NOTE: Use DEPC‐treated water ( appendix 2A) to prepare all solutions.

Support Protocol 5: Synthesis of DNA Probes by Primer Extension of Double‐Stranded Plasmid or PCR Product in a Thermal Cycler Using Thermostable Polymerase

  Materials
  • 0.1 to 10 pmol/µl oligonucleotide to be labeled
  • 150 mCi/ml [γ‐32P]ATP (7000 Ci/mmol)
  • 10× T4 polynucleotide kinase buffer (see recipe)
  • 10 U/µl T4 polynucleotide kinase
  • Gel loading buffer ( appendix 2A)
  • 20% denaturing polyacrylamide gel
  • 5 M ammonium acetate
  • 100% ethanol
  • RNase‐free microcentrifuge tubes (e.g., Ambion)
  • 95°C heating block
  • Additional reagents and equipment for removal of unincorporated nucleotides from probes by column chromatography (CPMB UNIT ) or ethanol precipitation (CPMB UNIT ) and gel purifying probes (see protocol 3)
CAUTION: Radioactive materials require special handling; all supernatants must be considered radioactive waste and disposed of appropriately.NOTE: Use DEPC‐treated water ( appendix 2A) to prepare all solutions.
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Figures

Videos

Literature Cited

Literature Cited
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   Berk, A.J. and Sharp, P.A. 1977. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease‐digested hybrids. Cell 12:721‐732.
   Brown, P.C., Thorgeirsson, S.S., and Silverman, J.A. 1993. Cloning and regulation of the rat mdr2 gene. Nucl. Acids Res. 21:3885‐3891.
   Burczynski, M.E., Lin, H.K., and Penning, T.M. 1999. Isoform‐specific induction of a human aldo‐keto reductase by polycyclic aromatic hydrocarbons (PAHs), electrophiles, and oxidative stress: Implications for the alternative pathway of PAH activation catalyzed by human dihydrodiol dehydrogenase. Cancer Res. 59:607‐614.
   Burton, E.A., Tinsley, J.M., Holzfeind, P.J., Rodrigues, N.R., and Davies, K.E. 1999. A second promoter provides an alternative target for therapeutic up‐regulation of utrophin in Duchenne muscular dystrophy. Proc. Natl. Acad. Sci. U.S.A. 96:14025‐14030.
   Chomczynski, P. 1992. Solubilization in formamide protects RNA from degradation. Nucl. Acids Res. 20:3791.
   Lee, J.J. and Costlow, N.A. 1987. A molecular titration assay to measure transcript prevalence levels. Methods Enzymol. 152:633‐648.
   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.
   Myers, R.M., Larin, Z., and Maniatis, T. 1985. Detection of single‐base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science 230:1242‐1246.
   Schenborn, E.T. and Mierendorf, R.C. 1985. A novel transcription property of SP6 and T7 RNA polymerases: Dependence on template structure. Nucl. Acids Res. 13:6223‐6236.
   Tran, P., Leclerc, D., Chan, M., Pai, A., Hiou‐Tim, F., Wu, Q., Goyette, P., Artigas, C., Milos, R., and Rozen, R. 2002. Multiple transcription start sites and alternative splicing in the methylenetetrahydrofolate reductase gene result in two enzyme isoforms. Mamm. Genome 13:483‐492.
   Turnbow, M.A. and Garner, C.W. 1993. Ribonuclease protection assay: Use of biotinylated probes for the detection of two messenger RNAs. BioTechniques 15:267‐270.
   Wundrack, I. and Dooley, S. 1992. Nonradioactive ribonuclease protection analysis using digoxygenin labeling and chemiluminescent detection. Electrophoresis 13:637‐638.
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