RNA Detection

3.2 Imaging RNA in
Fixed Cells by Using
CuAAC Reaction

1. After transfecting AMUTP for 1 h, remove the transfection
   mix. Wash the cells in each well three times with 1 mL of 1
   PBS (seeNote 11).


2. Add freshly prepared 1 mL of 3.7% formaldehyde in 1PBS to
   each well and incubate for 15 min at RT.


3. Remove the fixative and wash the cells in each well twice with
   1mLof1PBS.


4. Add 1 mL of Triton X 0.5% solution to each well and further
   incubate the cells at RT for 15 min.


5. Meantime prepare fresh CuAAC reaction mix.


6. Remove the Triton X 0.5% solution (step 4), then wash the
   cells in each well with 1 mL of 1PBS (35 min). Remove
   the wash solution.


7. Add 500μL of freshly prepared CuAAC reaction mix to each
   well. Swirl the plate gently to insure even distribution of reac-
   tion mix over the coverslip.


8. Incubate the plate for 30 min at RT in dark.


9. Remove the reaction cocktail and wash the cells with 1 mL of
   wash buffer (1PBS containing 2 mM sodium azide) for
   20 min and rinse again with 1 mL of 1PBS.


10. Counter-stain the DNA by using DAPI (500μL, 2μg/mL) in
    dark for 2 min at RT. Wash the coverslips with 1 mL of 1PBS
    (seeNote 8).


11. Remove the coverslip and place it upside-down on a micro-
    scope slide containing 10μL of SlowFade Gold Antifade
    Mountant (Invitrogen). Seal the edges of coverslip using nail
    polish.


12. Image the cells using a confocal laser scanning microscope with
    an oil immersion objective at 40or 63(Fig.3,seeNotes 5
    and 12 ).

3.3 Imaging RNA in
Fixed Cells by Using
SPAAC Reaction

1. Perform the steps as enumerated in Subheading3.1 (steps
   1 – 6 ) and3.2 (steps 1– 4 ).


2. Prepare a fresh cyclooctyne dye solution and transfer it to the
   coverslip.


3. Swirl the plate gently to insure even distribution of reaction
   mix over the coverslip.


4. Incubate the plate for 2 h at RT in dark.


5. Wash cells once with 1 mL of Triton X 0.01% solution. Further,
   wash the cells with 1 mL of 1PBS for 5 min. Repeat this
   washing step with 1PBS two more times.


6. Counter-stain DNA with DAPI and image the cells by follow-
   ing thesteps 9– 12 described in Subheading3.2 (Fig.4).

Imaging Newly Transcribed RNA in Cells 365