RNA Detection

2.3 Reagents for
Azide–Alkyne
Cycloaddition Reaction

1. 7.5 mM Alexa594 alkyne: Prepare a stock solution of Alexa
   Fluor®594 alkyne (7.5 mM) by adding 69μL of DMSO to
   0.5 mg Alexa Fluor® 594 (Thermo Scientific). Vortex the
   solution, spin and store at
   40 C(seeNote 6).


2. 10 mM Cy3 DBCO: Prepare a primary stock solution of
   20 mM Cy3 DBCO by adding 101μL of DMSO to 2 mg
   Cy3 DBCO vial (Click Chemistry Tools). Mix the content well
   by pipetting and vortexing and prepare 50μL working stock of
   10 mM Cy3 DBCO in DMSO. Store Cy3 DBCO stock solu-
   tions at
   40 C(seeNote 6).


3. 100 mM CuSO 4 : Freshly prepare the CuSO 4 solution (1 mL)
   using autoclaved water. Store this solution at 4C(seeNote 7).


4. 100 mM sodium ascorbate: Freshly prepare the sodium ascor-
   bate solution by dissolving 19.81 mg of sodium ascorbate in
   1 mL of autoclaved water. Store sodium ascorbate solution at
   4 C(seeNote 7).


5. CuAAC reaction mix (per coverslip): 429μL TBS buffer, 20μL
   100 mM CuSO 4 ,1μL 7.5 mM Alexa594 alkyne, 50μL
   100 mM sodium ascorbate. Prepare freshly by adding ingredi-
   ents in the indicated order atstep 5of Subheading3.2.


6. Cyclooctyne dye solution (per coverslip): 0.5μL 10 mM Cy3
   DBCO in 500μL1PBS containing 0.01% Triton X. Prepare
   freshly atstep 2of Subheading3.3.


7. Triton X 0.01% solution: 0.01% Triton-X-100 in 1PBS.


8. Washing buffer: 1PBS containing 2 mM sodium azide.

2.4 DNA Staining
Dyes

1. 1 mg/mL DAPI: Prepare DAPI stock solution using 1PBS
   in an amber-colored 1.5 mL Eppendorf tube. Mix well by
   pipetting and vortexing until DAPI dissolves completely.
   Store this stock solution at 4C in the dark. Prepare 500μL
   2 μg/mL working solution for staining DNA freshly by dilut-
   ing DAPI stock 1:500 in 1PBS (seeNote 8).


2. 20 mM Hoechst 33342: Store Hoechst 33342 stock solution
   at
   20 C. Thaw the vial at RT and freshly prepare 1μM stock
   solution of Hoechst dye (1 mL) in the complete DMEM by
   serial dilution (seeNote 9).

3 Methods

Reagents, buffers, media, and labware such as pipettes, plastic vials,

and Falcons necessary for cell culture experiments should be ster-

ilized by wiping with 70% aqueous ethanol and then only trans-

ferred to the cell culture hood. A flowchart of steps involved in the

imaging of cellular RNA by using azide–alkyne cycloaddition reac-

tion is provided in Fig.2.

Imaging Newly Transcribed RNA in Cells 363