RNA constructs and in vitro transcription.

This protocol describes generation of purified in vitro transcribed RNA, starting from template generation. It was based on the proprietary protocol used in the paper and adapted to make use of commercially available components.

1.    Amplify and purify DNA template (encoding T7 promoter, transcription start site, 5’ and 3’UTRs, coding sequence and polyA-tail) according to the instructions of the Plasmid Midi kit

2.    Sequence plasmid DNA to ensure correct sequence and integrity of the polyA tail

3.    Linearize 10 µg plasmid DNA with a restriction enzyme cutting only at the end of the encoded RNA sequence directly after the poly-A tail:

·         10 µg    plasmid DNA

·         2 µL      restriction buffer

·         10-20 U restriction enzyme

·         To 20 µL Nuclease-free Water

4.    Mix thoroughly and incubate reaction mix at 37°C for 2-4 hours

5.    Stop the reaction by adding 1 µL 0.5 M EDTA, 2 µL 3 M NaOAc and 40 µL 100% (v/v) EtOH, mix and chill at -20°C for 1h.

6.    Centrifuge at 4°C for 15 min at maximum speed to pellet the DNA.

7.    Dissolve DNA pellet in 10 µL nuclease-free water

8. Run aliquot of linearized DNA template on an 1% (w/v) agarose TAE gel to ensure efficiency of linearization reaction and determine concentration via UV measurement

9. Assemble in vitro transcription reaction as described in the mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (Invitrogen, catalog number: AM1345). In our case, poly-A is plasmid encoded. No additional poly-A reaction is needed.

·         Briefly: Thaw the frozen reagents and keep on ice, except 10x T7 reaction buffer (store at room temperature). Assemble transcription reaction at room temperature

·   10 µL        T7 2-fold NTP/ARCA

·   2 µL          10-fold T7 Reaction Buffer

·   1 µg          linear template DNA

·   to 20 µL Nuclease-free Water

·   2 µL          T7 Enzyme Mix

·         Mix thoroughly and incubate reaction mix at 37°C

·         After 1 hour of incubation, add TURBO DNase 1 and incubate for additional 15 min at 37°C

10.  Purify RNA via Lithium chloride precipitation method

·         Add 50 µL of Lithium Chloride Precipitation Solution and mix thoroughly.

·         Chill for ≥30 min at –20°C.

·         Centrifuge at 4°C for 15 min at maximum speed to pellet the RNA.

·         Remove supernatant and wash pellet with ~1 mL 70% (v/v) ethanol

·         Centrifuge at 4°C for 15 min at maximum speed

·         Remove 70% (v/v) ethanol, and resuspend the RNA in RNase-free water or 10 mM HEPES/ 0.1 mM EDTA buffer

11.  Determine the RNA concentration via UV measurement. Yield should be in the range of 10-30 µg RNA / 20 µL IVT reaction.

12.  Check RNA integrity by loading 80 ng on a 1% TAE agarose gel

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