Synthesis of fluorescent mRNA 

Source: Simon Bullock


  1. Prepare linearized template for sense RNA
  2. Set up the following reaction: total volume 25 µl
  3. reaction mix:
    template DNA (2-5 µg)
    2,5 µl 10xTranscription buffer*
    0.3 mM 7mG(5')pppG cap analogue
      4. 1 µl NTP mix
      0.04 mM fluorescent UTP**
      1 µl RNase Inhibitor, 20 units (Stratagene or Roche)
      0.5 µl SP6, T3 or T7 RNA polymerase, 25 units (Roche)
      DEPC-H2O

  4. Incubate at 37°C, 2 h
  5. Add 5 units RNase-free DNaseI, Incubate 37°C, 10 min
  6. Make up to 75 µl with RNase-free dH2O and extract once with phenol/chloroform/IAA (some unincorporated UTP is extracted too)
  7. Spin aqueous phase through an RNA-grade G50 column to remove unincorporated nucleotides, reduces background
  8. Precipitate the RNA with NH4OAc/EtOH and dissolve in suitable volume of dH2O or injection buffer. We usually get a yield of 4-10 µg of RNA and load a tenth or twentieth of this into the needle (diluted in injection buffer). This protocol typically results in 1 fluorophore/200-250 nucleotides
  9. Check quality by running 1/10-1/20 of product on a gel and take OD reading

Notes:

NTP mix: (prepare as 12,5x or 25x, Roche)
0.4mM ATP
0.4mM CTP
0.36mM UTP
0.12mM GTP

cap (mGpppG) thaw completely in 37°C

template DNA: (similar as for normal RNAsynthesis)
cut 5-10 µg of DNA in 100 µl volume for 3-4 h. then extract with phenol:chloroform:IAA and with chloroform, precipitate with 0,1 vol 3M NaOAc and 2,5 vol ethanol, wash with 70% EtOH, dry in speed vac and dissolve in 10 µl of DEPC treated water, store at -20°C

*Roche buffer contains DTT. If using other buffers, one may need to add this.
**Fluorescent UTPs are:    
-Alexa 488- or Alexa 546-UTP
(Molecular Probes, expensive but very bright and photostable)
-Cy3- or Cy5-UTP    
(from New England Nuclear, much cheaper but less bright).
Fluorescein UTP-labelled RNAs are, for some reason, not active in our assay (presumably because this label could interfere with RNA structure)