Materials and Reagents

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

Equipment

Illustra^TM MicroSpin^TM G-25 column (GE Healthcare, model: 27-5325-01 )

Procedure

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.
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

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

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.