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5′-End Labeling of RNA with [γ-32P]ATP and T4 Polynucleotide Kinase Donald C. Rio Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080739 Email Alerting Service Subject Categories
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Protocol
5′-End Labeling of RNA with [γ-32P]ATP and T4 Polynucleotide Kinase Donald C. Rio
This protocol uses T4 polynucleotide kinase to catalyze the transfer of a radiolabeled, terminal (γ) phosphate of ATP to the 5′ -hydroxyl terminus of a DNA or RNA molecule. The reaction is very efficient and hence is used as a general method for phosphorylating polynucleotides or oligonucleotides.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
EDTA (0.5 M; pH 8.0) Ethanol (100%) [γ-32P]ATP (>7000 Ci/mmol; 167 mCi/mL) RNA sample of interest (20 pmol of 5′ ends) The RNA molecules must have free 5′ -hydroxyl termini. If the sample was prepared by in vitro transcription and the RNA molecules have 5′ phosphate or 5′ triphosphate groups, the sample must be treated with calf intestine alkaline phosphatase to remove the 5′ phosphate(s) before rephosphorylation with T4 polynucleotide kinase. Also, if the RNA has been precipitated with ammonium acetate, it is important to make sure that any ammonium (NH4+) ions are removed because any free NH4+ ions will inhibit T4 polynucleotide kinase.
SDS extraction buffer Sodium acetate (3 M; pH 5.2) T4 kinase buffer (10×) T4 polynucleotide kinase (10 units/μL) (e.g., OptiKinase from Affymetrix) Equipment
Dry ice/ethanol bath Incubator or water bath (37˚C) Microcentrifuge Microcentrifuge tubes Pasteur pipette Adapted from RNA: A Laboratory Manual, by Donald C. Rio, Manuel Ares Jr., Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080739
441
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CHICAGO LIBRARY on October 13, 2014 - Published by Cold Spring Harbor Laboratory Press
D.C. Rio
Sephadex G-25 gel filtration spin columns SpeedVac METHOD
1. Set up a kinase reaction with RNA in the following order in a microcentrifuge tube: 20 pmol of 5′ ends 15 µL 2 µL 2 µL (100 pmol)
RNA sample of interest H2O T4 kinase buffer (10×) [γ-32P]ATP (>7000 Ci/mmol; 167 mCi/mL) T4 polynucleotide kinase (10 units/μL)
2 µL
2. Incubate the reaction for 1 h at 37˚C.
3. Stop the reaction by adding 2 µL of 0.5 M EDTA (pH 8.0).
4. Remove unincorporated radioactivity using a gel filtration spin column. 5. Precipitate the RNA with ethanol. i. Dilute the RNA to 200 µL with SDS extraction buffer and add sodium acetate to a final concentration of 0.3 M. ii. Add three volumes of cold 100% ethanol. iii. Chill in a dry ice/ethanol bath for at least 10 min. iv. Centrifuge at maximum speed in a microcentrifuge for 10 min. v. Aspirate the supernatant with a Pasteur pipette. Add 100 µL of cold 100% ethanol to the pellet and repeat Steps 5.iii–iv. vi. Discard the supernatant. Air-dry the RNA pellet or briefly dry the pellet in a SpeedVac. 6. Resuspend the RNA pellet in 10 µL of H2O. Store for up to 2 wk at −20˚C or −80˚C. RELATED INFORMATION
This protocol can be used to radiolabel the 5′ ends of RNA molecules for protein footprinting, gel-shift analyses (see Electrophoretic Mobility Shift Assays for RNA-Protein Complexes [Rio 2014]), chemical probing or protection (see RNA Structure Determination Using Nuclease Digestion [Nilsen 2013] and RNA Structure Determination Using Chemical Methods [Caprara 2013]), chemical sequencing, and sequencing by specific RNase digestions. RECIPES T4 Kinase Buffer (10×)
Reagent Tris-HCl (1 M, pH 7.5) MgCl2 (1 M) Dithiothreitol (DTT) (0.5 M) H2O
Quantity (for 1 mL)
Final concentration (10×)
500 µL 100 µL 100 µL
0.5 M 0.1 M 50 mM
300 µL
Store for up to 6 mo at −20˚C. 442
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080739
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CHICAGO LIBRARY on October 13, 2014 - Published by Cold Spring Harbor Laboratory Press
5’ End Labeling of RNA
SDS Extraction Buffer
Reagent
Quantity (for 500 mL)
Final concentration
10 mL 1 mL 25 mL 464 mL
20 mM 1 mM 0.5% (w/v) –
Tris–HCl (1 M, pH 7.5) EDTA (0.5 M, pH 8.0) SDS (10% w/v) H2O Store indefinitely at room temperature.
ACKNOWLEDGMENTS
For background information on this labeling reaction, see Chaconas and van de Sande (1980), Conway and Wickens (1989), Harrison and Zimmerman (1986), and Richardson (1981). REFERENCES Caprara M. 2013. RNA structure determination using chemical methods. Cold Spring Harb Protoc doi: 10.1101/pdb.prot078485. Chaconas G, van de Sande JH. 1980. 5′ -32P labeling of RNA and DNA restriction fragments. Methods Enzymol 65: 75–88. Conway L, Wickens M. 1989. Modification interference analysis of reactions using RNA substrates. Methods Enzymol 189: 369–379. Harrison B, Zimmerman SB. 1986. T4 polynucleotide kinase: Macromolecular crowding increases the efficiency of reaction at DNA termini. Anal Biochem 158: 307–315.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080739
Nilsen TW. 2013. RNA structure determination using nuclease digestion. Cold Spring Harb Protoc doi: 10.1101/pdb.prot072330. Richardson CC. 1981. Bacteriophage T4 polynucleotide kinase. In The enzymes (ed. Boyer PD), Vol. 14A, pp. 299–314. Academic, NY. Rio DC. 2014. Electrophoretic mobility shift assays for RNA-protein complexes. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080721.
443
5′-End Labeling of RNA with [γ-32P]ATP and T4 Polynucleotide Kinase Donald C. Rio Cold Spring Harb Protoc; doi: 10.1101/pdb.prot080739 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Probes (64 articles) Radiolabeled Probes (35 articles) RNA (218 articles) RNA, general (180 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CHICAGO LIBRARY on October 13, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
5′-End Labeling of RNA with [γ-32P]ATP and T4 Polynucleotide Kinase Donald C. Rio
This protocol uses T4 polynucleotide kinase to catalyze the transfer of a radiolabeled, terminal (γ) phosphate of ATP to the 5′ -hydroxyl terminus of a DNA or RNA molecule. The reaction is very efficient and hence is used as a general method for phosphorylating polynucleotides or oligonucleotides.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
EDTA (0.5 M; pH 8.0) Ethanol (100%) [γ-32P]ATP (>7000 Ci/mmol; 167 mCi/mL) RNA sample of interest (20 pmol of 5′ ends) The RNA molecules must have free 5′ -hydroxyl termini. If the sample was prepared by in vitro transcription and the RNA molecules have 5′ phosphate or 5′ triphosphate groups, the sample must be treated with calf intestine alkaline phosphatase to remove the 5′ phosphate(s) before rephosphorylation with T4 polynucleotide kinase. Also, if the RNA has been precipitated with ammonium acetate, it is important to make sure that any ammonium (NH4+) ions are removed because any free NH4+ ions will inhibit T4 polynucleotide kinase.
SDS extraction buffer Sodium acetate (3 M; pH 5.2) T4 kinase buffer (10×) T4 polynucleotide kinase (10 units/μL) (e.g., OptiKinase from Affymetrix) Equipment
Dry ice/ethanol bath Incubator or water bath (37˚C) Microcentrifuge Microcentrifuge tubes Pasteur pipette Adapted from RNA: A Laboratory Manual, by Donald C. Rio, Manuel Ares Jr., Gregory J. Hannon, and Timothy W. Nilsen. CSHL Press, Cold Spring Harbor, NY, USA, 2011. © 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080739
441
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CHICAGO LIBRARY on October 13, 2014 - Published by Cold Spring Harbor Laboratory Press
D.C. Rio
Sephadex G-25 gel filtration spin columns SpeedVac METHOD
1. Set up a kinase reaction with RNA in the following order in a microcentrifuge tube: 20 pmol of 5′ ends 15 µL 2 µL 2 µL (100 pmol)
RNA sample of interest H2O T4 kinase buffer (10×) [γ-32P]ATP (>7000 Ci/mmol; 167 mCi/mL) T4 polynucleotide kinase (10 units/μL)
2 µL
2. Incubate the reaction for 1 h at 37˚C.
3. Stop the reaction by adding 2 µL of 0.5 M EDTA (pH 8.0).
4. Remove unincorporated radioactivity using a gel filtration spin column. 5. Precipitate the RNA with ethanol. i. Dilute the RNA to 200 µL with SDS extraction buffer and add sodium acetate to a final concentration of 0.3 M. ii. Add three volumes of cold 100% ethanol. iii. Chill in a dry ice/ethanol bath for at least 10 min. iv. Centrifuge at maximum speed in a microcentrifuge for 10 min. v. Aspirate the supernatant with a Pasteur pipette. Add 100 µL of cold 100% ethanol to the pellet and repeat Steps 5.iii–iv. vi. Discard the supernatant. Air-dry the RNA pellet or briefly dry the pellet in a SpeedVac. 6. Resuspend the RNA pellet in 10 µL of H2O. Store for up to 2 wk at −20˚C or −80˚C. RELATED INFORMATION
This protocol can be used to radiolabel the 5′ ends of RNA molecules for protein footprinting, gel-shift analyses (see Electrophoretic Mobility Shift Assays for RNA-Protein Complexes [Rio 2014]), chemical probing or protection (see RNA Structure Determination Using Nuclease Digestion [Nilsen 2013] and RNA Structure Determination Using Chemical Methods [Caprara 2013]), chemical sequencing, and sequencing by specific RNase digestions. RECIPES T4 Kinase Buffer (10×)
Reagent Tris-HCl (1 M, pH 7.5) MgCl2 (1 M) Dithiothreitol (DTT) (0.5 M) H2O
Quantity (for 1 mL)
Final concentration (10×)
500 µL 100 µL 100 µL
0.5 M 0.1 M 50 mM
300 µL
Store for up to 6 mo at −20˚C. 442
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080739
Downloaded from http://cshprotocols.cshlp.org/ at UNIV OF CHICAGO LIBRARY on October 13, 2014 - Published by Cold Spring Harbor Laboratory Press
5’ End Labeling of RNA
SDS Extraction Buffer
Reagent
Quantity (for 500 mL)
Final concentration
10 mL 1 mL 25 mL 464 mL
20 mM 1 mM 0.5% (w/v) –
Tris–HCl (1 M, pH 7.5) EDTA (0.5 M, pH 8.0) SDS (10% w/v) H2O Store indefinitely at room temperature.
ACKNOWLEDGMENTS
For background information on this labeling reaction, see Chaconas and van de Sande (1980), Conway and Wickens (1989), Harrison and Zimmerman (1986), and Richardson (1981). REFERENCES Caprara M. 2013. RNA structure determination using chemical methods. Cold Spring Harb Protoc doi: 10.1101/pdb.prot078485. Chaconas G, van de Sande JH. 1980. 5′ -32P labeling of RNA and DNA restriction fragments. Methods Enzymol 65: 75–88. Conway L, Wickens M. 1989. Modification interference analysis of reactions using RNA substrates. Methods Enzymol 189: 369–379. Harrison B, Zimmerman SB. 1986. T4 polynucleotide kinase: Macromolecular crowding increases the efficiency of reaction at DNA termini. Anal Biochem 158: 307–315.
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot080739
Nilsen TW. 2013. RNA structure determination using nuclease digestion. Cold Spring Harb Protoc doi: 10.1101/pdb.prot072330. Richardson CC. 1981. Bacteriophage T4 polynucleotide kinase. In The enzymes (ed. Boyer PD), Vol. 14A, pp. 299–314. Academic, NY. Rio DC. 2014. Electrophoretic mobility shift assays for RNA-protein complexes. Cold Spring Harb Protoc doi: 10.1101/pdb.prot080721.
443
