RNA Detection

(BamHI/ClaI) cassettes can be individually cloned into

reporter constructs. Conventional PCR amplification of the

PP7 and MS2 cassettes described here is not recommended

due to the repetitiveness of the DNA sequences. A co-

localization control plasmid (seeSubheading3.4.3) can be

generated by introduction of a stop codon in front of the PP7

cassette using site-directed mutagenesis.

2. Seed 30,000 HeLa cells stably expressing NLS-MCP-Halo,
   NLS-PCP-GFP and the TRICK reporter construct in 2 mL
   DMEM + 10% FBS + 1% Pen/Strep into a 35 mm imaging dish
   ~48 h prior to the experiment. Take care that cells are homo-
   geneously distributed within the dish to improve cytoplasmic
   imaging of mRNAs (seeNote 1).


3. Incubate cells for 2 days at 37C and 5% CO 2. Longer or
   shorter incubation times are possible depending on the cell line
   being used.


4. Immediately prior to the experiment, prewarm PBS and Fluor-
   obrite™DMEM + 10% FBS to 37C.


5. Halo-label cells by incubation with 1 mL 100 nM JF 549 in
   DMEM + 10% FBS for 30 min at 37C and 5% CO 2.


6. Remove medium from cells, wash three times with PBS and
   add prewarmed Fluorobrite™DMEM + 10% FBS + 1μg/mL
   doxycycline to induce TRICK reporter expression.


7. Since TRICK measures the first round of translation, it is
   important to consider the timing of induction and the acquisi-
   tion of images. For the HeLa cell line we use, TRICK tran-
   scripts begin to enter the cytoplasm approximately 45 min after
   doxycycline addition.

3.2 Image
Acquisition

1. Equilibrate the incubation chamber of the microscope to 37C
   and 5% CO 2.


2. Align cameras prior to image acquisition using a multicolor
   calibration slide.


3. Identify suitable cells for image acquisition using the red laser
   (MCP-Halo channel) at low intensities to reduce photobleach-
   ing prior to the experiment. Choose cells with well-resolved
   (high signal/noise) diffraction-limited mRNA particles (diam-
   eter approximately 4 pixels or 0.36μm for optical settings
   described above) at densities that facilitate SPT.


4. Image cells in both channels simultaneously using laser powers,
   camera gain and exposure time compatible with SPT. Exposure
   times should be no longer than 50 ms to ensure that fast
   moving particles can be unambiguously tracked between
   subsequent frames. Laser power should be adapted to yield
   maximum signal intensity while limiting bleaching the samples,

TRICK Assay 377