RNA Analysis by Nuclease Protection

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

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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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 ³² P‐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 H₂ O ( 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‐³² P]UTP or [α‐³² P]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 H₂ O ( 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 H₂ O ( 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 )

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 H₂ O ( appendix 2A )
Liquid nitrogen or dry ice/methanol bath
10× primer‐extension buffer (see recipe )
10 mCi/ml [α‐³² P]dATP or [α‐³² P]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 )

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‐³² P]dATP or [α‐³² P]dCTP (3000 Ci/mmol)
2 U/µl Taq or Tth DNA polymerase
DEPC‐treated H₂ O ( 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 )

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‐³² P]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 )

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Figures

Figure 5.1.1 RNase protection assay of AU‐rich target sequence using different RNases. Sea urchin total RNA (5 µg) was hybridized in replicate reactions to a probe for an AT‐rich cyclin gene. Reactions were treated with either RNase T1, RNase A, RNase A/RNase T1 mixture, or RNase I (all from E. coli ), using two‐fold different amounts of each RNase. AU‐rich regions are especially susceptible to overdigestion. Several discrete bands and a high background of diffuse bands are seen with all conditions except the reactions containing RNase T1 alone, which show most of the signal in a single protected fragment. Because RNase T1 cleaves only after G residues, there is little nonspecific cleavage at transiently separated AU‐rich strands. The size of the degraded unhybridized probe fragments in the samples treated with RNase T1 alone is slightly larger than in other lanes, but no full‐length probe remains in the experimental or control lanes where T1 alone was used.

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Figure 5.1.2 Typical RNase protection assay using a probe for mouse β‐actin. A 300‐base mouse β‐actin probe was hybridized to two‐fold‐increasing amounts of mouse total‐liver RNA (from 0.625 to 10 µg; lanes 4 to 8) and analyzed by RNase protection. The intensity of the protected fragment increases with increasing sample RNA, and the size of the protected fragment (250 bases) reflects a downward size shift from the full‐length probe due to degradation of the vector‐transcribed sequences in the probe. (When probes are made from PCR templates, the full‐length probe and protected fragment may be exactly the same size.) The absence of signal in the yeast RNA control lane treated with RNase (lane 3) shows that the amount of RNase used was sufficient to completely degrade all unhybridized probe. The mock‐digested yeast RNA control (lane 2) shows that in the absence of added RNase, the probe was intact. The figure also shows the effect of altering the amount of RNase A/RNase T1 mixture used to digest unhybridized probe in reactions containing 10 µg of total mouse liver RNA. Amounts of RNase A/RNase T1 mixture used to digest unhybridized probe in each reaction were as follows, relative to the standard amount of 5 µg/ml RNase A/100 U/ml RNase T1: lane 10, 0.001×; lane 11, 0.01×; lane 12, 0.1×; lane 13, 1×; lane 14, 3×; lane 15, 10×; lane 16, 33×. Note the wide range of effective RNase concentrations for this probe‐target combination. Although the optimal RNase A/RNase T1 concentration to use in an RNase protection assay may vary for different probes, it will usually fall within the range from 0.1× to 2× the standard amount. RNase I, a single‐strand‐specific RNase isolated from E. coli , is sometimes used for RNase protection assays; however the effective dynamic range is not as wide as for RNase A/RNase T1. Use of RNase I generally requires that enzyme concentration be optimized separately for different amounts of total RNA used in the hybridization reaction.

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Figure 5.1.3 Synthesis of single‐stranded (ss) DNA probes by primer extension. (A ) Primer extension from DNA sequences cloned into pT7/T3. The plasmid is first linearized with a restriction enzyme at the opposite end of the insert from that used for priming. A specific primer oligonucleotide (at the 5′ side of the insert) designed to hybridize to a specific sequence in the plasmid is added, then the DNA is heat denatured. The reaction is snap frozen, thawed, and put on ice, then incubated with a master mix containing salts, dNTPs, radiolabel, and Klenow fragment. The resulting ssDNA probe is then gel‐purified. (B ) Primer extension from double‐stranded PCR products. A specific nested primer oligonucleotide designed to hybridize to a specific sequence on the PCR product is added, then the resulting ssDNA probe is generated and purified as described above.

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Figure 5.1.4 Single‐stranded DNA probes prior to gel purification. (A ) A 340‐base antisense probe synthesized with Klenow fragment of DNA polymerase by primer extension from a plasmid template using the Prime‐A‐Probe kit (Ambion). (B ) The same probe synthesized by primer extension in a thermal cycler using thermostable polymerase. (C ) A 25‐base oligonucleotide probe end‐labeled with polynucleotide kinase using the Kinase Max kit (Ambion).

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Figure 5.1.5 5′ end mapping of mRNA transcription start sites.

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Figure 5.1.6 Multiprobe time‐course RNase protection assay using three probes. Probes for mdr1b and mdr2 (genes involved in multiple drug resistance during chemotherapy in cancer treatment) were designed to span the regions of highest variability between the two target mRNAs. These genes are difficult to distinguish on northern blots because of their similar size and 72% homology. The regions chosen for the mdr probes contained numerous sites of multiple‐base mismatch (three or more bases) with each other, to assure that the probes were specific for the intended targets (i.e., multiple‐base mismatches are cleaved efficiently by RNase A). The probe for the constitutively expressed glyceraldehyde‐6‐phosphate dehydrogenase ( GAPDH ) gene was included as an internal standard to normalize the amount of total RNA used in each sample. Lanes 2 through 6 show a time course (½, 1, 2, 3, and 4 days) for induction of mdr1b and mdr2 by experimental treatment with carbon tetrachloride in rat total‐liver RNA; lane 1 shows the probes hybridized to 10 µg normal (i.e., untreated with carbon tetrachloride) rat liver. Note the variable time course for the expression of the two mdr genes. Data obtained using the RPA II kit (Ambion); reproduced from Brown et al., (National Cancer Institute, NIH).

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Figure 5.1.7 Typical S1 nuclease protection assay using a single‐stranded antisense DNA probe for mouse β‐actin. Target RNA consists of decreasing amounts of mouse‐liver total RNA (10 µg, 5 µg, 2.5 µg, and 0.5 µg; lanes 4 through 8). Note the linear increase in intensity of the protected fragment with increasing sample RNA and the downward size shift of the full‐length probe at ∼340 bases to the protected fragment at 250 bases. The size shift is due to digestion of the region of the probe that contains nonhomologous vector sequences from the primer‐extension reaction. Lanes 2 and 3 contain probe plus 10 µg yeast RNA, treated with S1 nuclease (lane 3), or mock‐digested (lane 2).

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Videos

Literature Cited

Literature Cited
	Benkusky, N.A., Fergus, D.J., Zucchero, T.M., and England, S.K. 2000. Regulation of the Ca2+‐sensitive domains of the maxi‐K channel in the mouse myometrium during gestation. J. Biol. Chem. 275:27712‐27719.
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	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.
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RNA Analysis by Nuclease Protection

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited