MicroRNA Amplification and Recognition through Locked-nucleic-acid In situ Hybridization as a Novel Detection and Quantification Method

The scope of our research was to develop and validate an assay that simultaneously detects and quantifies microRNAs and multiple proteins within FFPE tissue all the better to spatially observe the relationship between both within the TME. It actually overcomes common limitations of existing molecular sequencing and traditional-ish by providing a direct quantification, colocalization, and simultaneous spatial detection of microRNAs and proteins, all without destroying the tissue and preserving cell and subcellular localization. This technology potentially paves the way for both basic research into regulatory networks, disease mechanisms, and translational or clinical studies seeking better biomarkers and therapeutic targets.

Begin by taking out formalin-fixed paraffin-embedded samples of human ovarian tumor, kept overnight at 60 degrees Celsius. Using RNAse-free water, clean all equipment and tools thoroughly. Next to perform deparaffinization, immerse the samples in xylene three times for five minutes each.

Rehydrate the tissues with 10 rounds of sequential diluted ethanol series, each for three minutes. And then, wash the samples once in TBS for two minutes. Apply a 3%hydrogen peroxide solution to the samples for 10 minutes.

Wash the samples twice for two minutes each with TBS. Then, apply a 3.7%paraformaldehyde solution for 10 minutes. After that, begin the first round of antigen retrieval using the easy retriever infrared system.

Submerge the slides in fresh EZ-AR 1 buffer, and microwave at 95 degrees Celsius for 15 minutes. Then rinse the samples in TBS for two minutes, followed by two two minute washes in TBST. To begin, draw a barrier around each tissue section using a hydrophobic barrier pen.

Add antibody blocking solution and incubate the slides for 30 minutes at room temperature in a humidity chamber. Now, apply the primary antibody diluted in the same blocking solution to fully cover the tissue section and incubate for one hour at room temperature in a humidity chamber. After washing the slides three times in TBST, add the anti-mouse and rabbit horseradish peroxidase solution to cover the tissue and incubate in the humidity chamber at room temperature for 10 minutes.

Now, dilute the first fluoro-4 at a 1:100 ratio with amplification dilutant. Apply this mixture to the tissue and incubate for 10 minutes at room temperature in a humidity chamber, avoiding direct light exposure. After performing the second round of antigen retrieval and blocking as shown previously, dilute the second primary antibody with the same blocking solution.

Apply the diluted antibody to cover the entire tissue section and incubate overnight for at least 16 hours at four degrees Celsius inside a humidity chamber. The next day, wash the slides three times for two minutes each in TBST and incubate with anti-mouse and rabbit horseradish peroxidase solution as shown earlier. Then, apply the diluted second fluoro-4 to the slides and incubate for 10 minutes at room temperature in the humidity chamber.

For probe application, wash the slides three times for two minutes each in TBST. To begin the third and final antigen retrieval, submerge the slides in EZ-AR 1 Elegance buffer, and microwave at 107 degrees Celsius for 15 minutes. Cool the slides at room temperature for 20 minutes before washing them once for three minutes in TBS.

Apply enough RNAscope Protease Plus to cover the tissue area and incubate at 40 degrees Celsius for 30 minutes using an oven. Then, heat the microRNA locked nucleic acid probe to 90 degrees Celsius for four minutes to denature it. Immediately dilute the denatured probe with microRNA in situ hybridization buffer.

After vortexing and spinning the prepared probe, add it to cover the entire tissue area and incubate for one hour at 60 degrees Celsius in the oven. Wash the slides once for five minutes in 5x SSC Buffer. Perform five stringent washes at 60 degrees Celsius using SSC Buffer for five minutes each.

Then, wash the slides once for five minutes in 0.2 times SSC at room temperature. To prepare the blocking buffer, combine TBS and 0.5%blocking reagent powder. Apply the blocking buffer and incubate for 30 minutes at room temperature in a humidified chamber.

Next, apply anti-digoxin and mouse horseradish peroxidase conjugate, and incubate for 30 minutes at room temperature in a humidified chamber. After three TBST washes, apply 1:50 dilution of the tyramide signal amplification plus cyanine 3 to the slides for 10 minutes at room temperature in a humidity chamber. Wash the slides three more times for five minutes each in TBST.

Apply enough DAPI working solution diluted 1:1000 in TBS to fully cover the tissue section. Incubate the slides for five minutes at room temperature in a humidified chamber with ambient humidity. Wash the slides three times for five minutes each in TBST.

Apply cover slips using mounting media and let the slides dry for 10 minutes at room temperature. Store the dried slides at four degrees Celsius in the dark for 24 to 72 hours before imaging or up to three months for longer term storage. The miRNA expression of miR-181c-3p was consistently detected in both control and antibody stained slides, with visibly stronger signals concentrated in the tumor cell regions.

Application of the anti-EpCAM antibody with a 620 nanometer emission fluorophore stained the tumor cells in the HGSC sample. Application of the anti-CD8 antibody with a 620 nanometer emission fluorophores distinctly marked CD8 positive T-cell. Combined protein and miRNA detection enabled spatial localization of miR-181c-3p relative to EpCAM positive tumor cells and CD8 positive immune cells.

The positive control probe targeting U6 small nuclear RNA produced strong fluorescent signals across all tissue types, confirming probe effectiveness and tissue integrity. The negative control probe which used a scrambled miRNA sequence showed no detectable signal in any tissue type, indicating no non-specific binding. The target probe for miR-181c-3p showed strong expression in the high-grade serous carcinoma tissue with no signal in normal ovarian tissue or normal fallopian tube tissue, demonstrating the specificity and accuracy of the probe and assay.