Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

This method can address key research questions in neuroscience, oncology, and beyond, such as cell type specific expression and spatial mapping of splice variants within the brain and tumor microenvironments. The main advantage of this technique is that it can detect exon junctions, short sequences, and point mutations in the tissue context with very high sensitivity and specificity in single cells. Demonstrating the procedure will be Hell Pimentel, an associate scientist from our laboratory.

To prepare the tissue, fix it immediately following dissection in 10%neutral buffered formalin at room temperature for 16 to 32 hours. Subsequently, the sample is processed and embedded in paraffin using standard procedures. Cut the embedded tissue into five plus or minus one micron sections using a microtome.

Mount the sections on electrostatically adhesive glass slides and air dry at room temperature over night. One hour before performing the RNA NC2 hybridization assay, place the mounted tissue slides in a slide rack and bake in a circulating air oven at 60 degrees Celsius. Prior to starting the RNA NC2 hybridization assay, ensure the hybridization oven is set to 40 degrees Celsius.

Thoroughly wet the humidifying paper, remove any residual water, and place the lid onto the humidity control tray. Prepare 200 milliliters of commercially available 1X target retrieval reagent by adding 20 milliliters of 10X target retrieval reagent to 180 milliliters of water. Place two slide holders in a steamer.

Fill one slide holder with 200 milliliters of 1X target retrieval reagent and the other with 200 milliliters of water. Using the steamer, heat both solutions to a boil. Warm the 50X wash buffer to 40 degrees Celsius for 10 to 20 minutes.

Then prepare three liters of 1X wash buffer by diluting 60 milliliters of pre-warmed 50X wash buffer with 2.94 liters of water. In a fume hood, prepare 50%of Gill's hematoxylin counterstaining solution by adding 100 milliliters of Gill's hematoxylin to 100 milliliters of water. Prepare bluing reagent by adding 1.43 milliliters of 20 to 30%ammonium hydroxide to 250 milliliters of water.

Pre-warm the target probes at 40 degrees Celsius for 10 minutes prior to probe hybridization. Bring the seven amplifying reagents, Amp 0 to 6, to room temperature. Start the sample depariffinization by removing sections from the oven and immersing the mounted tissue slides in xylene with agitation for five minutes.

Depariffinize again in fresh xylene for five minutes. Dehydrate in 100%ethanol with agitation for two minutes. And repeat again in fresh 100%ethanol for two minutes.

Air dry the slides at 60 degrees Celsius in a circulating air oven for five minutes until they are completely dry. Next, incubate the sections with about four drops of ready-to-use hydrogen peroxide at room temperature for 10 minutes to quench the endogenous peroxidase activity. Decant the solution from the slides and rinse them twice with water.

Briefly dip the sections in the pre-warmed water and then place the sections in 200 milliliters of the target retrieval reagent at 100 degrees Celsius in the steamer for 15 to 30 minutes. Remove slides from the target retrieval and rinse twice in water. Dip in 100%ethanol for three minutes.

Then dry the slides at 60 degrees Celsius in a circulating air oven until completely dry. Once the slides are dry, use a hydrophobic pen to draw an approximately 0.75 by 0.75 inch hydrophobic barrier around the section. Let the barrier dry completely at room temperature for one minute.

Place the slides in a slide holder and place the slide holder in the pre-warmed humidity controlled tray. Add about four drops of protease 3 to each slide and incubate at 40 degrees Celsius in the hybridization oven for 15 to 30 minutes. Decant the solution from the slides and rinse them twice with water.

To begin the probe hybridization procedure, add about four drops of the appropriate ready-to-use probe solution to cover the entire section. Hybridize the probes at 40 degrees Celsius in the hybridization oven for two hours. Decant the solution from the slides and wash the slides in 200 milliliters of 1X wash buffer at room temperature with occasional agitation for two minutes.

Repeat the washing procedure. The next step is to amplify the signal by incubating the sections in a series of amplifying reagents. Add about four drops of Amp 0 to cover the entire section on each slide and incubate at 40 degrees Celsius in the hybridization oven for 30 minutes.

Then decant the solution and wash the slides in 200 milliliters of 1X wash buffer at room temperature with occasional agitation for two minutes. Repeat the washing procedure. In the same manner, incubate the sections in Amp 1 through Amp 6 at the temperature and for the amount of time indicated in this schematic.

After the slides have been treated with the series of amplification reagents, prepare a Fast Red dye working solution. Add the appropriate volume of Fast Red-B dye and Fast Red-A dye into a tube and mix well. Decant the excess liquid from the slides and add the Fast Red dye working solution to the slides.

Incubate the slides in a tray with a cover at room temperature for 10 minutes. After 10 minutes, decant the Fast Red solution and rinse the slides twice by dipping up and down with tap water. To counterstain the tissue sections, immerse the slides in 50%Gill's hematoxylin solution at room temperature for two minutes.

Wash the slides with tap water, repeating this several times until the slides are clear while the sections remain purple. Dip the slides into 02%ammonia water two to three times for bluing. Replace the ammonia water with tap water and wash the slides three to five times.

Dry the slides in a circulating air oven at 60 degrees Celsius for 15 minutes until completely dry. Place one to two drops of mounting reagent on a cover slip and place the slide over the cover slip while avoid any trapping of air bubbles. Air dry the slides for at least five minutes.

Lastly, observe the slides under a standard bright field microscope. Double-Z exon junction probes were designed for determining the EGFRvIII status in two formalin fixed paraffin embedded, or FFPE glioblastoma samples. Both wild type probes detected signal in both samples, indicating that the two samples express wild type EGFR.

However, the mutant probe only detected signal in the EGFRvIII positive sample and not in the EGFRvIII negative sample. Positive and negative control probes were included in the assay. When a short target assay was performed on Jurkat cells, prepared as an FFPE cell pellet using antisense or sense probes, targeting the sequences in red, robust staining was observed with antisense probes for both CDR3-alpha and CDR3-beta, whereas sense probes detected little to no signal.

dapB was used as a negative control. The assay is also able to detect point mutations. In this example, two probes were designed, one to detect the L858R mutated EGFR sequence and another to detect the EGFR L858 wild type sequence.

The mutant probe detected signal in the H1975 cell line, which is heterozygous for the EGFR L858R mutation, but did not detect signal in the H2229 cell line, which only expresses EGFR L858 wild type. The wild type probe detected signal in both cell lines. While attempting this procedure, it's important to remember to properly fix your tissues, run positive and negative control probes, and use the slide processing system in hybridization oven.

Following this procedure, other methods like IHC with cell markers can be performed in order to answer additional questions like identifying cell type specific expression of splice variants or short target sequences. After its development, this technique paved the way for researchers in neuroscience, oncology, and beyond to explore alternative splicing events in the tissue context, identifying spatially mapped point mutations, and visualize highly homologous and short sequences in situ. Don't forget that working with neutral buffered formalin and xylene can be extremely hazardous and precautions such as working in a fume hood and wearing personal protective equipment should always be taken while performing this procedure.