Poly A RNA in situ protocol

Protocol for in situ for total polyA mRNA localization in metazoan cell lines

General Notes

this protocol has been used with Drosophila S2R+ cells, as well as a variety of mammalian cell types including HeLa, COS7, U2OS, N1E and N2a (mouse neuroblastomas). It really should work with any cell line. You may counterstain the cells with an antibody after the hybridization step (details are included in the protocol). This protocol is NOT recommended for detection of specific mRNA transcripts, because the probe is short and directly labeled with the fluorophore, so for a specific message the probe length would not be optimal and the signal would be too weak. This protocol works with cells grown on glass coverslips or chamber slides, as well as in 96- or 384- well clear bottom plates. If using plates as opposed to coverslips or slides, it is recommended that you centrifuge the plate at 1000-1200 rpm for 1-2 minutes immediately before each aspiration step to minimize cell loss. If signal is less than optimal for your particular cell type or application, experiment with the probe dilution (range of 1:100-1:1500), hybridization temperature (try range of 37 – 50 degrees), and wash conditions (heating the wash solutions to the hybridization temp can reduce background). ALL STEPS AND WASHES ARE PERFORMED AT ROOM TEMP UNLESS OTHERWISE SPECIFIED.

Buffers to make (all DEPC-treated):

20X SSC (for diluting for other buffers)

2X SSC

2X SSC + 0.1%TX-100

4X SSC

1.0M Tris pH8.0

1X PBS

4%paraformaldehyde in PBS, pH 7.4 (make this fresh, OR make a batch and aliquot and store at -20 degrees. It should be good for up to one year when frozen.)

Hybridization Buffer:

Yeast tRNA - 1mg/mL

BSA - 0.005%

Dextran sulfate - 10%

Formamide, deionized - 25%

20X SSC + DEPC water so that final buffer volume is in 2X SSC

Fixation and Hybridization

- Aspirate the media from each well and rinse once with PBS.

- Aspirate the PBS and add 4% paraformaldehyde (made in 1x PBS, pH7.4) to each well. Let the cells fix for 10 minutes

- Aspirate the paraformaldehyde and add 100% COLD methanol to each well for 10 minutes (methanol helps to permeabilize the nucleus)

- Aspirate the methanol and add 70% ethanol to each well for at least 10 minutes. The ethanol step helps to rehydrate the cells after the fixation steps. Plates can safely be stored for several days at 4 degrees in ethanol before proceeding with the hybridization step without any loss of mRNA signal or compromise in cell morphology.

- Aspirate the ethanol and add 1M Tris pH8.0 to each well for 5 minutes.

- While the cells are incubating in Tris, add the probe to the appropriate amount of hybridization buffer needed for the number of samples you have. The probe I use is a 1ug/uL stock of 5’-labeled Cy3-Oligo-dT(30). This can be ordered from most major oligo suppliers. Dilute this stock 1:1000 in hybridization buffer for a final concentration of 1ng/uL. Also note, other fluorophores, like Cy2 (green) or Cy5 (far red) could be used for probe labeling if desired.

- Aspirate the Tris, and add hybridization buffer with probe to each well

- Seal the plate/slides/coverslips in a plastic bag with a couple of wet kimwipes to maintain humidity

- Incubate at 37 degrees for at least one hour. Keep the samples covered from here forward, so as not to bleach the flourophore. Hybridization can continue overnight if desired.

Washing and Visualization

- After hybridization, remove the plate from the bag and wash samples once with 4x SSC.

- Wash again with 2x SSC

- IF YOU WISH TO STAIN WITH AN ANTIBODY: dilute your primary antibody in 2xSSC + 0.1% triton-X-100 and incubate for 1 hr at RT (no need to block, as hyb solution contains BSA). Wash three times with 2x SSC, then incubate with your secondary antibody plus DAPI or Hoechst in 2xSSC + 0.1% triton-X-100.

- IF YOU ARE NOT STAINING WITH AN ANTIBODY: Incubate in 2xSSC + 0.1% triton-X-100 with Hoechst dye or DAPI at 1:1000 for 15 minutes, to stain the DNA

- After staining (with antibody or just Hoechst) Wash twice with 2x SSC

- IF USING PLATES: Leave 25-100uL of 2x SSC in each well. Seal the plates well. The plate is now ready for imaging. mRNA signal is stable and detectable for two weeks or longer when stored in 2x SSC at 4 degrees.

- IF USING COVERSLIPS OR SLIDES: remove the coverslip, dry excess SSC with a kinwipe, and mount on a slide with appropropriate mounting media. The fluorescent signal will last a month or longer. Morphology should be preserved indefinitely.