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5’ end-labeled RNA molecules are useful substrates to analyse the endo- and exonucleolytic activities of various ribonucleases. Here two protocols are given to synthesize P32 labeled RNAs with a 5’ PPP or 5’ P moiety. 5’ exoribonucleases generally do not work on 5’ PPP RNA and require a 5’ P substrate. The activity of certain endoribonucleases like Escherichia coli (E. coli) RNase E or Bacillus subtilis (B. subtilis) RNase Y can be stimulated by a 5’ P moiety.

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Synthesis of 5’ end-labeled RNA

Molecular Biology > RNA > RNA synthesis
Author: Harald Putzer
Harald PutzerAffiliation: CNRS FRE3630, Institut de Biologie Physico-Chimique, Paris, France
For correspondence: putzer@ibpc.fr
Bio-protocol author page: a68
Vol 2, Iss 14, 7/20/2012, 5305 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.241

[Abstract] 5’ end-labeled RNA molecules are useful substrates to analyse the endo- and exonucleolytic activities of various ribonucleases. Here two protocols are given to synthesize P32 labeled RNAs with a 5’ PPP or 5’ P moiety. 5’ exoribonucleases generally do not work on 5’ PPP RNA and require a 5’ P substrate. The activity of certain endoribonucleases like Escherichia coli (E. coli) RNase E or Bacillus subtilis (B. subtilis) RNase Y can be stimulated by a 5’ P moiety.

Keywords: RNA Labeling, RNA Synthesis, Radioactive labeling

Materials and Reagents

  1. RQ1 DNase (Promega Corporation, catalog number: M6101 )
  2. Calf intestinal phosphatase (F. Hoffmann-La Roche, catalog number:  713023 )
  3. Phenol/Chlorophorm (1:1) (MP Biomedicals, catalog number: AQUAPH01 )/ 
    (Carlo Erba, catalog number: 438601 )
  4. T7 RNA polymérase (Promega Corporation, catalog number: P207B )
  5. T4 Polynucleotide Kinase (Biolabs, catalog number: M0201 )
  6. RNasin RNase inhibitor (Promega Corporation, catalog number: N2611 )
  7. DTT (Promega Corporation, catalog number: P117B )
  8. NTPs (F. Hoffmann-La Roche, catalog number: set1277057 )
  9. γ32P GTP (6,000 Ci/mmole, 10 μC/μl) (PerkinElmer, catalog number: BLU504Z )
  10. γ32P ATP (3,000 Ci/mmole, 10 μCi/μl) (PerkinElmer, catalog number: BLU502A )
  11. 3 M NaOAc (pH 4.7) (see Recipes) 

Equipment

  1. IllustraTM MicroSpinTM G-25 column (GE Healthcare, model: 27-5325-01 )

Procedure

  1. Synthesis of 5’-P RNA  
    1. In vitro Transcription (30 μl)
      T7 RNA polymerase 5x buffer           6 μl
      RNasin (40 U/μl)                                0.75 μl
      100 mM DTT                                      3 μl
      2.5 mM NTPs                                     6 μl
      DNA template (PCR fragment)          400 ng
      H2O (RNase free)                              to final volume (30 μl)
      T7 RNA polymerase (20 U/μl)           1.5 μl
      1. Incubation 1 h 30 min at 37 °C.
      2. Add to the reaction mix 2 μl of RQ1 DNase (1 U/μl).
      3. Incubation 30 min at 37 °C (digestion of template DNA).
      4. Put on ice to stop reaction.
      5. Addition of 18 μl H2O to bring volume to 50 μl.
      6. Purification of RNA by Spin-G25 column (load sample on gel bed, centrifuge 2 min at 735 x g (~3,000 rpm in a microfuge).
    2. Dephosphorylation step (50 μl)
      Calf intestinal phosphatase (CIP 1 U/μl)                    2.5 μl
      CIP 10x buffer                                                            5 μl
      Purified RNA                                                              32.5 μl
      H2O                                                                            10 μl
      1. Incubation 30 min at 37 °C.
      2. Add 1 vol of Phenol/Chlorophorm (1:1), vortex 3 min.
      3. Centrifuge 3 min at >10,000 x g to separate phases.
      4. Precipitate upper aqueous phase by adding 1/10 vol. 3 M NaOAc (pH 4.7) + 3 vol. ETOH (96 %).
      5. Centrifuge 10 min at >10,000 x g, take off EtOH and wash pellet once with 70% EtOH by inverting tube.
      6. Air-dry pellet and take up the RNA in the desired volume of H2O.
    3. Labelling reaction (20 μl)
      Dephosphorylated RNA                                            1 μg
      T4 Polynucleotide Kinase 10x buffer                         2 μl
      RNase inhibitor (40 U/μl)                                           0.25 μl
      γ32P ATP (10 μCi/μl)                                                  5 μl
      T4 Polynucleotide Kinase (10 U/μl)                           1.25 μl
      H2O                                                                            to 20 μl final volume
      1. Incubate 2 h at 37 °C.
      2. Precipitation as in step 2 and take up the pellet in desired volume.

  2. Synthesis of 5’-PPP RNA
    T7 RNA polymerase 5x buffer                       6 μl
    RNasin (40 U/μl)                                            0.75 μl
    DTT (100 mM)                                               3 μl
    ATP, UTP, CTP (2.5 mM)                               6 μl
    GTP (100 μM)                                                3.75 μl
    PCR                                                               400 ng
    γ32P GTP                                                       7.5 μl
    H2O                                                                to 30 μl final volume
    T7 RNA polymerase (20 U/μl)                       1.6 μl
    1. Incubate 1 h, 30 min at 37 °C.
    2. Add to the reaction mix 2 μl of RQ1 DNase (1 U/μl).
    3. Incubation 30 min at 37 °C (digestion of DNA).
    4. Precipitation with 1/10 vol 3 M NaOAc (pH 4.7) + 3 vol EtOH + 25 μg Glycogen.
    5. Centrifuge 10 min at >10,000 x g, take off EtOH and wash pellet once with 70% EtOH by inverting tube.
    6. Air-dry pellet and take up the RNA in the desired volume of H2O.

Recipes

  1. 3 M NaOAc (pH 4.7)
    Dissolve 40.8 g of sodium acetate. 3H2O in a final volume of 100 ml, adjust pH with glacial acetic acid.

Acknowledgments

This laboratory protocol is a free adaption of various published and unpublished protocols and has evolved over time. We acknowledge the support by funds from the CNRS (UPR 9073) and Univ Paris Diderot, Sorbonne Paris Cite.

References

  1. Taverniti, V., Forti, F., Ghisotti, D. and Putzer, H. (2011). Mycobacterium smegmatis RNase J is a 5'-3' exo-/endoribonuclease and both RNase J and RNase E are involved in ribosomal RNA maturation. Mol Microbiol 82(5): 1260-1276.


How to cite: Putzer, H. (2012). Synthesis of 5’ end-labeled RNA. Bio-protocol 2(14): e241. DOI: 10.21769/BioProtoc.241; Full Text



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10/24/2013 1:17:53 AM  

Marco Binder
Dept. Infectious Diseases, Medical Faculty Heidelberg

After reading the material requirements in the top, it appears to me that one step simply has been forgotten in the protocol, which would be the phosphorylation of the CIPped RNA by polynucleotide kinase and gamma-P32-ATP. Then everything would make sense again!

:-)

10/24/2013 2:54:45 AM  

Harald Putzer (Author)
CNRS FRE3630,Institut de Biologie Physico-Chimique

Dear Marco,
you are correct, the labeling step is not in protocol even though it was in the version that I had submitted. Sorry for that. The missing part is:
3) Labelling reaction (20 µl)

Dephosphorylated RNA 1 µg
T4 Polynucleotide Kinase buffer 10X 2 µl
RNase inhibitor (40 U/µl) 0.25 µl
?32P ATP (10 µCi/µl) 5 µl?
T4 Polynucleotide Kinase (10U/µl) 1.25 µl
H2O to 20 µl final volume

a. incubate 2 hours at 37°C
b. Precipitation as in step (2) and take up the pellet in desired volume

I will ask to correct this on the web site.

Harald

10/24/2013 10:50:43 PM  

Bio-protocol Editorial Team
bio-protocol.org

Dear Dr. Putzer and Macro,

The missing step has been added as shown at step 1c. We deeply apologize for our mistake and any confusion that have caused to you.

Sincerely,
Bio-protocol editing team

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10/24/2013 12:31:19 AM  

Marco Binder
Dept. Infectious Diseases, Medical Faculty Heidelberg

I do not understand, how CIP treatment would generate a 5'-monophosphate; from what I understand, CIP removes ALL phosphoryl-groups from DNA, RNA and protein...

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