Synthesis and Labeling of RNA In Vitro

Chao Huang1, Yi‐Tao Yu2

1 Process Science Downstream, Bristol‐Myers Squibb Company, East Syracuse, New York, 2 Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, New York
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 4.15
DOI:  10.1002/0471142727.mb0415s102
Online Posting Date:  April, 2013
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Abstract

This unit discusses several methods for generating large amounts of uniformly labeled, end‐labeled, and site‐specifically labeled RNAs in vitro. The methods involve a number of experimental procedures, including RNA transcription, 5′ dephosphorylation and rephosphorylation, 3′ terminal nucleotide addition (via ligation), site‐specific RNase H cleavage directed by 2′‐O‐methyl RNA‐DNA chimeras, and 2‐piece splint ligation. The applications of these RNA radiolabeling approaches are also discussed. Curr. Protoc. Mol. Biol. 102:4.15.1–4.15.14. © 2013 by John Wiley & Sons, Inc.

Keywords: in vitro transcription; radiolabeled RNAs; RNA research

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro Synthesis of Uniformly Radiolabeled RNAs
  • Basic Protocol 2: In Vitro Synthesis of 5′ End‐Radiolabeled RNAs
  • Basic Protocol 3: In Vitro Synthesis of 3′ End‐Radiolabeled RNAs
  • Basic Protocol 4: In Vitro Synthesis of Site‐Specifically Radiolabeled RNAs
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vitro Synthesis of Uniformly Radiolabeled RNAs

  Materials
  • 5× buffer for transcription (see recipe)
  • 3 NTP mix (see recipe)
  • 100 µM CTP (Thermo Scientific)
  • 10 µCi/µl [α32P]CTP (sp. act. 800 Ci/mmol; PerkinElmer)
  • 1 µg/µl DNA template (from linearized plasmid, PCR, or oligodeoxynucleotides)
  • 40 U/µl RNase inhibitor (Thermo Scientific)
  • 20 U/µl T7 RNA polymerase (Thermo Scientific)
  • DNase I (RNase‐free; Thermo Scientific)
  • G50 buffer (see recipe)
  • 25:24:1 phenol/chloroform/isoamyl alcohol
  • 100% ethanol
  • Additional reagents and equipment for urea‐PAGE (Williams and Chaput, ) and autoradiography ( appendix 3A)

Basic Protocol 2: In Vitro Synthesis of 5′ End‐Radiolabeled RNAs

  Materials
  • 10× buffer for CIP (see recipe)
  • RNA substrate from in vitro transcription ( protocol 1) or purified directly from cells (endogenous RNA; Huang and Yu, )
  • 40 U/µl RNase inhibitor (Thermo Scientific)
  • 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific)
  • G50 buffer (see recipe)
  • 10× buffer for T4 PNK forward reaction (see recipe)
  • 10 µCi/µl [γ32P]ATP (3000 Ci/mmol; PerkinElmer)
  • 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific)
  • Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA ( protocol 1, steps 4 to 9), urea‐PAGE (Williams and Chaput, ), autoradiography ( appendix 3A), and “freeze‐thaw” elution/ethanol precipitation ( protocol 1, steps 10 to 13)

Basic Protocol 3: In Vitro Synthesis of 3′ End‐Radiolabeled RNAs

  Materials
  • 10× buffer for T4 RNA ligase (see recipe)
  • 10 mM ATP (Thermo Scientific)
  • RNA substrate with 3′ hydroxyl end derived from in vitro transcription ( protocol 1) or purified directly from cells (endogenous RNA; Huang and Yu, )
  • 5′ 10 µCi/µl [32P]pCp (3000 Ci/mmol; PerkinElmer)
  • 10 U/µl T4 RNA ligase (Thermo Scientific)
  • G50 buffer (see recipe)
  • Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA ( protocol 1, steps 4 to 9), urea‐PAGE (Williams and Chaput, ), autoradiography ( appendix 3A), and “freeze‐thaw” elution/ethanol precipitation ( protocol 1, steps 10 to 13)

Basic Protocol 4: In Vitro Synthesis of Site‐Specifically Radiolabeled RNAs

  Materials
  • 200 pmol/µl 2′‐O‐methyl RNA‐DNA chimera (Integrated DNA Technologies, Inc.)
  • RNA substrate from in vitro transcription ( protocol 1) or in vivo purification
  • 10× buffer for RNase H (see recipe)
  • 2 U/µl RNase H (Amersham)
  • 40 U/µl RNase inhibitor (Thermo Scientific)
  • G50 buffer (see recipe)
  • 10× buffer for CIP (see recipe)
  • 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific)
  • 10× buffer for T4 PNK forward reaction (see recipe)
  • 32P]ATP (3000 Ci/mmol, 10 µCi/µl) (PerkinElmer)
  • 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific)
  • Bridging DNA oligo (Integrated DNA Technologies, Inc.)
  • 10× buffer for T4 DNA ligase (see recipe)
  • 5 U/µl T4 DNA ligase (Thermo Scientific)
  • 95°C heat block
  • Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA ( protocol 1, steps 4 to 9), urea‐PAGE (Williams and Chaput, ), autoradiography ( appendix 3A), and “freeze‐thaw” elution/ethanol precipitation ( protocol 1, steps 10 to 13)
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Figures

Videos

Literature Cited

Literature Cited
   Huang, C. and Yu, Y.T. 2010. Targeted 2′‐O methylation at a nucleotide within the pseudoknot of telomerase RNA reduces telomerase activity in vivo. Mol. Cell. Biol. 30:4368‐4378.
   Ma, X., Zhao, X., and Yu, Y.T. 2003. Pseudouridylation (Psi) of U2 snRNA in S. cerevisiae is catalyzed by an RNA‐independent mechanism. EMBO J. 22:1889‐1897.
   Ma, X., Yang, C., Alexandrov, A., Grayhack, E.J., Behm‐Ansmant, I., and Yu, Y.T. 2005. Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA‐guided or RNA‐independent mechanism. EMBO J. 24:2403‐2413.
   Milligan, J.F., Groebe, D.R., Witherell, G.W., and Uhlenbeck, O.C. 1987. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15:8783‐8798.
   Moon, K.H., Zhao, X., and Yu, Y.T. 2006. Pre‐mRNA splicing in the nuclei of Xenopus oocytes. Methods Mol. Biol. 322:149‐163.
   Romfo, C.M., Maroney, P.A., Wu, S., and Nilsen, T.W. 2001. 3′ splice site recognition in nematode trans‐splicing involves enhancer‐dependent recruitment of U2 snRNP. RNA 7:785‐792.
   Schenborn, E.T. and Mierendorf, R.C. Jr. 1985. A novel transcription property of SP6 and T7 RNA polymerases: Dependence on template structure. Nucleic Acids Res. 13:6223‐6236.
   Suydam, I.T. and Strobel, S.A. 2009. Nucleotide analog interference mapping. Methods Enzymol. 468:3‐30.
   Williams, B.A.R. and Chaput, J.C. 2010. Synthesis of peptide‐oligonucleotide conjugates using a heterobifunctional crosslinker. Curr. Protoc. Nucl. Acid Chem. 42:4.41.1‐4.41.20.
   Wu, S., Romfo, C.M., Nilsen, T.W., and Green, M.R. 1999. Functional recognition of the 3′ splice site AG by the splicing factor U2AF35. Nature 402:832‐835.
   Yu, Y.T. 1999. Construction of 4‐thiouridine site‐specifically substituted RNAs for cross‐linking studies. Methods 18:13‐21.
   Yu, Y.T. and Nilsen, T.W. 1992. Sequence requirements for maturation of the 5′ terminus of human 18 S rRNA in vitro. J. Biol. Chem. 267:9264‐9268.
   Yu, Y.T., Maroney, P.A., Darzynkiwicz, E., and Nilsen, T.W. 1995. U6 snRNA function in nuclear pre‐mRNA splicing: A phosphorothioate interference analysis of the U6 phosphate backbone. RNA 1:46‐54.
   Zhao, X. and Yu, Y.T. 2004. Detection and quantitation of RNA base modifications. RNA 10:996‐1002.
   Zhao, X., Li, Z.H., Terns, R.M., Terns, M.P., and Yu, Y.T. 2002. An H/ACA guide RNA directs U2 pseudouridylation at two different sites in the branchpoint recognition region in Xenopus oocytes. RNA 8:1515‐1525.
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