RNA Detection

3.1 Transfection of
AMUTP into Cells

1. Culture mammalian cells (e.g., HeLa cells) in complete
   DMEM supplemented with 10% FBS (seeNotes 1and 3 ).


2. Seed 0.3–0.5 million HeLa cells in a 6-well plate containing
   glass cover slips. Place the plate in the CO 2 incubator at 37C
   for 24 h (seeNote 10).


3. Take a new 6-well dish and add 1 mL of HBS to each well and
   transfer the coverslips with cells to the individual wells with the
   help of a forceps.


4. Remove HBS using aspirator and carefully add 600μL of the
   AMUTP transfection mix over the coverslip.


5. Gently swirl the plate in clockwise and anticlockwise direction
   so that the transfection mix covers the surface of the coverslip.
   Place the plate in the CO 2 incubator at 37C for 1 h (seeNote
   3 ).

Fig. 2Flowchart depicting the stepwise procedure for imaging newly transcribed RNA in cells by using AMUTP.
Azide-labeled RNA transcripts were detected by performing CuAAC/SPAAC reactions in fixed/live cells

364 Anupam A. Sawant et al.