• One day prior to transfection, plate HeLa cells in 12-well plate at approximately 50-70% density
• On the following day, use HeLa Monster Rgt. to transfect HeLa cells with 330 ngs of plasmid encoding the mRNA reporter of interest (plasmid should have Tet2 promoter), 330 ngs of plasmid encoding the tetracycline trans-activator protein, and 125ngs of plasmid encoding GFP-hDcp1a as a PB marker (might not be needed if not looking at PBs), and any other plasmid of interest. (Note: Remember to include tetracycline in transfection mix if you intend to pulse transcription of the mRNA reporter)
• One day after transfection, split cells to 8-well chamber slides: Depending on the density of the cells, split 50-100 uL of trypsinized cells (which should be resuspended in 1 mL of medium after trypsinization) into each well of the chamber slide. Bring the total volume in each well of the chamber slide up to 500 uL. (Remember to keep the cells in tetracycline at a conc. of 50ngs/mL if you do not intend to begin a transcriptional pulse at this time.)
• One day after splitting cells to chamber slides: Begin a transcriptional pulse by washing the cells in PBS and then adding media (-) tetracycline to each well of the chamber slide. The pulse should last about 8 hrs, but may vary depending on the experiment (This works well for ARE, siRNA, and NMD reporters).

Note: For all subsequent steps do not let the cells dry out between washings!

• After pulsing for the desired time, remove the media from each well and incubate the cells for >10mins in 100 uL of 4% paraformaldehyde (use “EM grade” in yellow cabinet).
• Remove the formaldehyde and wash the cells twice in PBS.
• Add 250uL of 70% “RNA only” ethanol. Cover chamber slide in tin foil and incubate O/N at 4 degrees C.

• On the following day, incubate cells for 10 mins at room temp. in In Situ Rehydration Buffer (see end of protocol). While this is incubating, prepare the in situ probe cocktail as follows (you’ll need to prepare 100 uL of this mix for each sample):

  50% formamide
  2X SSC
  0.02% RNase-free BSA
  10% dextran sulfate
  2mM vanadyl-ribonucleoside complexes
  40ug carrier RNA x number of samples
  15-20ng probe x number of samples

• Add this 100 uL of cocktail to each well and incubate the chamber slides (in tin foil) at 37 degrees C O/N.

• On the following day, wash cells twice with in situ rehydration buffer. Then add in situ rehydration buffer again to each well and incubate cells for 30 min at 37 degrees C. Repeat this last wash one more time.
• Wash one last time with water. Remove water, add vectashield to the cells and place a coverslip on top. Apply force to the cover slip in order to spread the vectashield to the outer corners of the slide. Seal with nail polish and view on fluorescent microscope
• In Situ Rehydration Buffer:

  50% formamide
  2X SSC
  ddH20