Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

The overall goal of this procedure is to investigate the dynamics of long non-coding exist, RNA, and its associated epigenetic modification during X chromosome inactivation by using a quick fluorescent in situ hybridization combined with immunofluorescence. This protocol requires cytosine preparation of slides with differentiating embryo body cells, which are permeated and then fixed for staining. Next fluorescent marker localized by an antibody to histone H three trimethyl lysine 27 accumulates on the inactive X chromosome.

The final step uses oligonucleotide probes that contain a five prime amino modification and are labeled with an AM mean reactive fluorophore to perform exist. RNA fish. Ultimately fluorescence microscopy is used to show the localization of exist RNA cloud and histone modification during E Es cells Differentiation.

The main advantage of this technique over existing methods is that we use multiple unique oligonucleotides, which are labeled with a single fluoro four. This provides us with trans specific detection, which reduces the background, and this also increases the penetration to the nucleus and it increases or enhances the intensity of the signals. In addition, when we increase the concentration of the probes, we can reduce significantly the time required for completing this immuno fish experiment.

Though this method can provide insight into the localization of exist RA on the inactive ex chromosome and it's associated chromo modification. It can be also applied to other biological systems for the detection of RAs and it's associated epigenetic modification, nuclear localization, and transgenerational regulation. To prepare the probes first, obtain multiple 20 to 30 nucleotide length oligos with a melting temperature of 63 to 65 degrees Celsius, and which contain a unique antisense sequence to an exist RNA and A five prime amino modification.

Suspend these oligos in pure water pool equimolar amounts of the various oligos for a total of 4.5 micrograms of DNA. Then following the manufacturer's instructions, label the oligos with an am mean reactive fluorescent dye following the labeling of the oligos. Pass them through a G 25 column.

Then using ethanol and sodium acetate, precipitate the oligos. This requires a half hour period at negative 80 degrees Celsius before a 21, 000 G spin at four degrees Celsius. Next, wash the pellet once with 70%ethanol after the wash.

Resus suspend the oligos in 50 microliters of nuclease free water or tris EDTA to a final concentration of 10 micromolar DNA. This makes the probe stock solution to use the probe reconcentrate it to 250 nano molar in hybridization buffer. To begin have ES or EB differentiating cells growing in six well dishes aspirate the culture medium from the wells and then wash the cells with room temperature.

PBS thoroughly aspirate the PBS. Next, add 0.5 milliliters of trypsin solution to each well and incubate the plate for five to 10 minutes. Stop the tripsin reaction by adding five milliliters of media to each well and gently pipetting them.

Then transfer the cells to a 15 milliliter tube. Now spin down the cells for five minutes at 200 Gs at room temperature, remove all the supernatant and resuspend the pallet of cells in two milliliters of PBS. Then count the cells and dilute the suspension to 400, 000 cells per milliliter in PBS.

Now, assemble each slide with a filter card, a chamber, and a clip. Make as many as needed and position them into the rotor of a cyto spin. Add 250 microliters of cell suspension to each slide and spin the slides at 1500 RPM for 10 minutes at room temperature.

Then perme and fix. Firstly, quickly transfer the slides to ice cold PBS for five minutes as a rinse step. Secondly, transfer the slides to ice cold CSK buffer for one minute to swell the thirdly, move the slides into CSKT buffer for five minutes to perme the cells.

Fourthly, wash the cells in ice cold CSK buffer for a minute. And lastly, fix them in room temperature 4%paraform aldehyde in PBS for 10 minutes. First, remove the slides from the PFA solution.

Then transfer them to a coplan jar with PBST for several seconds, followed by setting them on a slant to drain off. Next, load an empty pipette tip box with water below the tip holder insert. Then wipe off the back sides of the slides and transfer them to this box.

Now overlay each slide with 40 microliters of blocking solution and gently cover the slides with cover slips. Let them incubate in the box for 10 minutes on the bench. Then remove the cover slips and set the slides up on a slant.

Now add 40 microliters of the primary antibody solution. Replace the cover slips and let the slides incubate on the bench for 30 minutes after half an hour. Remove the unbound antibodies by washing the slides twice in PBST in coplan jars.

Use gentle shaking and let each wash go for five minutes. Then return the slides to a slant and let the PBST drain off. Follow by wiping off the excess solution from the slide backsides and then returning the slides to the box.

Now apply 40 microliters of one to 500 secondary antibody solution to each slide. Cover the slides with cover slips and let them incubate on the bench for 15 minutes protected from light going forward. The slides should always be protected from light.

Remove the antibodies using PBST as before, and then set the slides on a slant to drain off. When performing immunofluorescence, prewarm a box containing water to 37 degrees Celsius. After wiping off washing buffer from slide backsides, place them in a pipette tip box with temperature water and apply 500 microliters of fish wash buffer to each slide.

Let them incubate for five minutes. After five minutes, aspirate the solution from the slides next to each slide at eight microliters of the working probe at room temperature and apply a cover slip. Now transfer the slides to the prewarm slide box and incubate them for one to two hours at 37 degrees Celsius.

After the incubation, bathe the slides in fish wash buffer in a coplan jar at 37 degrees Celsius. When the cover slips fall off, which usually takes a few minutes, remove the cover slips and allow the slides to incubate for a five minutes. Then transfer the slides to a jar containing fish wash buffer with 0.5 nanograms of dappy stain per milliliter, also at 37 degrees Celsius.

Let the slides incubate in this solution for five minutes. Now wash the slide in two XSSC five minutes at 37 degrees Celsius. To finish up, set the slides on a slant drain excess two XSSC.

Wipe them off and mount them with four microliters of Antifa solution containing dpi. Quick immuno fish was used to observe the localization of exist RNA and histone modification. In differentiating female EB cells exist, RNA looks like a green cloud covering the entire length of the one inactivated X chromosome In these cells, the histone H three trimethyl lysine 27 modification is visualized in red and the nuclei are counterstain by DPI in blue.

Differentiating female mouse EB cells were processed at day 12 of differentiation. At this time, more than 90%of the cells showed an exist cloud in the cells Nuclei short oligonucleotide probes were efficiently able to penetrate resulting in the staining of almost all the histone H three trimethyl lysine 27 signals With exist RNA clouds in 150 nuclei, 97%of exist RNA signals were found to co localize with these methylated histones. Once mastered, this technique can be done in less than five hours to complete slight prepar preparation.

Immuno immuno fluorescence for his is three trimester ly in 27 and exist RA.If we performed properly, This immuno fish protocol can be applied to other RNA epigenetic modifications and proteins of interest. In order to apply this protocol to a different biological system, you might need some optimization. Good luck with your experiments.