Decapping eukaryotic mRNA with RppH (NEB #M0356)

Procedure

  1. Add in order:
    1. Nuclease-free water: 40 μl
    2. 10X NEB Thermopol buffer: 5 μl (1X= 20mM Tris-HCl, 10mM (NH4)2SO4, 10mM KCl, 2mM MgSO4, 0.1% Triton® X-100, pH 8.8@25°C)
    3. RNA up to 500 ng of RNA: 5 μl
    4. RppH (5,000 units/ml): 1 μl per 100 ng of RNA up 5 μl for 500 ng
    5. If using less than 5 μl RppH make volume of reaction up to 50 μl with water.
  2. Mix, pipette up and down a minimum of six times.
  3. Incubate for one hour at 37°C.
  4. Add 1 μl of 500mM EDTA. Pipette to mix.
  5. Stop the reaction by heating at 65°C for 5 minutes.
Notes: The above protocol can also be used for removing the 5´ triphosphate of in vitro transcripts to leave a 5´ monophosphate. This protocol requires the separate purchase of 10X Thermopol Buffer NEB #B9004S

Component 50 µl reaction
RNA 5 ng – 500 ng
10X Thermopol Buffer NEB (B9004S) 5 µl (10X)
RNA 5´ Pyrophosphohydrolase (RppH) (NEB #M0356S) 5 units per 5 ng RNA up to 25 units per 500ng of RNA
Nuclease-free Water To 50 µl
Incubation Time 1 hour*
Incubation Temperature 37°C

References:

Song, M. G., Bail, S., & Kiledjian, M. (2013). Multiple Nudix family proteins possess mRNA decapping activity. RNA. doi:10.1261/rna.037309.112

Neri F, Rapelli S, Krepelova A, Incarnato D, Parlato C, Basile G, et al. Intragenic DNA methylation prevents spurious transcription initiation. Nature. 2017;543:72–7.

Hetzel, Jonathan et al. “Nascent RNA Sequencing Reveals Distinct Features in Plant Transcription.” Proceedings of the National Academy of Sciences of the United States of America 113.43 (2016): 12316–12321. PMC. Web. 21 Mar. 2017.