RNA Detection

4 Notes

1. Thaw buffer solutions and media required for cell culture in the
   water bath (37C) containing autoclave water prior to use in
   cell culture experiments. Sterilize the bottles and Falcons by
   wiping with 70% ethanol, and then transfer them into the
   culture hood. Always open the reagents and media inside the
   cell culture hood only.


2. See manufacturer’s instruction for thawing FBS. We usually
   thaw the frozen stock of the heat inactivated FBS in +4C
   fridge and complete the thawing process at room temperature.
   FBS should be mixed few times during the thawing process by
   gently swirling the bottle. Aliquot 30–40 mL and store in deep
   freezer. Working aliquot of FBS can be also stored at 4C.
   Avoid vigorous shaking while handling FBS as it is susceptible
   to frothing.


3. The following RNA labeling protocol has been developed
   using HeLa cells with an optimized AMUTP concentration of
   1 mM and transfection time of 1 h. However, we have per-
   formed experiments to test a range of AMUTP concentrations
   (50μM–4.0 mM) and transfection time (15 min–3 h) to deter-
   mine the best labeling condition in HeLa cells. Apparently,
   longer incubation times (3 h) and concentrations of
   AMUTP2 mM resulted in detectable reduction in cellular
   RNA staining. In our experiments, DOTAP served as a signifi-
   cantly better transfecting agent for AMUTP as compared to
   Lipofectamine. Although this protocol will also work for other
   cell lines, it is recommended that the concentration of AMUTP
   and transfection time should be optimized for each of the cell
   lines.


4. Detergents are main constituents of Triton X-100 and exces-
   sive shaking will lead to frothing. Therefore, handle Triton X-
   100 carefully to avoid cross contamination during processing
   cells.


5. In order to confirm the specific incorporation of AMUTP into
   cellular RNA, we treat the cells with actinomycin D (RNA
   polymerase inhibitor). Essentially, the described procedure
   can be adopted wherein actinomycin D (final concentration
   2 μM, 600μL) prepared in Opti-MEM is added before trans-
   fecting AMUTP. The cells are incubated in CO 2 incubator at
   37 C for 5 h. Remove 150μL of actinomycin D solution and
   add freshly prepared 150μL of transfection mix and incubate
   for 1 h. Perform the fixation, permeabilization, and click stain-
   ing reaction as mentioned in Subheading3.2.


6. Thaw the vials at RT and spin it well prior to use. Store the
   stocks of fluorescent dyes in deep freezer (
   20 or
   40 C) and

Imaging Newly Transcribed RNA in Cells 369