Editing Capped mRNA (section)

=== In Vitro Transcription of Modified Cre RNA ===

13. This section is for the in vitro transcription of modified Cre mRNA using the above stated protocol. Begin by preparing a nucleotide triphosphate (NTP) mix. This mix recipe is sufficient for 5.4 reactions and this proportion of Cap:GTP allows for ~80% of transcripts to be capped, though this can contribute to reduced yield. The cap powder should be dissolved in 35 μL H2O. The recipe is as follows:<br/>Mix Concentration Desired Final Concentration<br/>Cap 15 mM 6 mM<br/>GTP 3.75 mM 1.5 mM<br/>ATP 18.75 mM 7.5 mM<br/>5methylCTP 18.75 mM 7.5 mM<br/>PseudoUTP 18.75 mM 7.5 mM

14. For a 2x reaction (40 μL), combine reagents in the following order in a PCR strip at room temperature. The NTP Mix and template should be thawed on ice, then moved to room temperature (but only for setting up the IVT, immediately return to ice afterward). The 10x Buffer can be pre-warmed to 37 °C. It is important to ensure there is no precipitate in the 10x Buffer. The template concentration should be pre-adjusted at 100 ng/μl.<br/>Custom NTP Mix 16 μL<br/>10x Buffer (Kit) 4 μL<br/>Template (Tail PCR) 16 μL (1.6 μg)<br/>T7 Enzyme Mix (Kit) 4 μL<br/>Total 40 μL

15. Incubate the reaction at 37 °C for up to 12 hours.

16. Perform DNase treatment with 2 μL TURBO DNase (Kit) for each reaction. Gently mix and incubate at 37 °C for 15 min.

17. Perform purification with MEGAclear Kit as described in the manufacturer’s instructions: Begin by pre-warming the 115 μL Elution Solution per reaction at 95 °C using PCR machine.

18. Bring the in vitro transcription (IVT) reaction volume to 100 μL by adding 58 μL of Elution Solution. Mix by pipetting and transfer the total volume to an RNase-free Eppendorf tube.

19. To the new RNase-free Eppendorf tube, add 350 μL Binding Solution Concentrate and mix by pipetting.

20. Add another 250 μL of 100% ethanol solution and mix by pipetting.

21. Transfer the sample to a filter cartridge (assembled on a collection tube).

22. Centrifuge for 1 min at 10,000 RPM. Take care to avoid the escape of glass fibers out of the column and into the collection tube. Discard the flow-through.

23. Wash each reaction with 500 μL Wash Solution and centrifuge for 1 min at 10,000 RPM. Discard the flow-through.

24. Repeat wash with 500 μL Wash Solution. Discard the flow-through.

25. Dry by centrifuging for 30 seconds at 10,000 RPM.

26. Place filter on a new collection tube and elute RNA by adding 50 μL of 95 °C Elution Buffer. Centrifuge for 1 min at 10,000 RPM.

27. Repeat the elution step with 50 μL of 95 °C Elution Buffer. Centrifuge for 1 minute at 10,000 RPM.

28. Quantify the RNA using a Nanodrop machine before proceeding to Antarctic phosphatase treatment and a second of purification by the MEGAclear Kit.

29. Perform Antarctic Phosphatase treatment by adding 10 μL 10x buffer to each reaction. Then add 3 μL Antarctic Phosphatase. Gently mix and incubate the reaction for 30 minutes at 37 °C. Optional: Inactivate reaction at for 5 minutes at 65 °C.

31. Perform RNA Purification with MEGAclear Kit as described above (Step 17 – 27).

32. Quantify the RNA using Nanodrop. Each 2x reaction will generally produce ~50 μg RNA (the expected yield of a 40 μL reaction is generally 40 – 60 μg).

33. Adjust the RNA concentration to 100 ng/μL using RNase-free TE buffer.

34. Aliquot RNA and store at −20 °C or −80 °C. −20 °C is preferable to reduce freeze/thaw cycles.

Note: To quality control RNA, load the RNA and run gel electrophoresis under native conditions. High quality RNA should result in the observation of a single band.