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

microRNAs
Although microRNAs (miRNA) are incredibly small, ranging 19-25 nucleotides in length, these single-stranded RNA molecules are potent regulators in gene expression, modulating messenger RNAs (mRNAs) post-transcriptionally through sequence-specific interactions¹. In essence, miRNAs act as negative regulators by binding to target mRNAs, either preventing their translation or promoting their degradation through mRNA cleavage, depending on the degree of complementarity, which ultimately leads to decreased protein output from the targeted gene. While miRNAs are basically non-coding RNA, approximately 40% of all miRNAs are still found within introns or exons of protein-coding genes². The functional purpose of miRNAs encompasses several key biological processes such as cell proliferation, angiogenesis, apoptosis, etc. Notably, the dysregulation of miRNAs has been implicated in tumorigenesis, as aberrant expression of specific miRNAs and miRNA-mediated gene regulation has been associated with oncogenic transformation in multiple cancer types³.

Since their discovery, miRNAs have been extensively researched in various diseases due to their tissue-specific expression and regulatory functions, with their dysregulation linked to many diseases, including neurodegenerative disorders, autoimmune disorders, and various cancers^4,5,6. The expression patterns of miRNAs can be correlated with cancer type, stage, and clinical variables, making miRNA profiling a valuable tool for cancer diagnosis and prognosis^4,5,6,7. The discovery of new miRNAs has also been accelerated by advancements in high-throughput sequencing technologies, enhancing the understanding of miRNA involvement in diverse biological processes⁸. The significance of miRNAs was underscored by the 2024 Nobel Prize in Medicine awarded to Victor Ambros and Gary Ruvkun for their discovery of the function of miRNA in posttranscriptional regulation⁹.

As valuable biomarkers for disease diagnosis and prognosis, the therapeutic potential of miRNAs has been explored, leading to the development of miRNA-targeting drugs. The therapeutic potential of miRNAs in cancer treatment lies in their ability to coordinately regulate modules of genes, potentially restoring normal cellular functions¹⁰. Previously, miRNA-based anticancer therapies have been developed either as monotherapy or in combination with existing targeted therapies to enhance treatment responses and increase cure rates¹¹. The dynamic spectrum of miRNAs has positioned them as essential resources for innovative cancer treatment strategies. In the context of therapeutic resistance, targeting miRNAs has shown promise in reducing recurrence and resistance to traditional cancer treatments, potentially improving patient survival by targeting cancer cells¹². By exploring the roles of miRNAs in modulating the activities of various cell types in the tumor microenvironment (TME), unique and clinically relevant cancer treatments can potentially be discovered.

Available assays to detect miRNAs
Currently, the most common method for miRNA measurement relies on extracted RNA for quantitative real-time PCR (q-RT-PCR) or Next Generation Sequencing (NGS), losing spatial context. There are companies that will provide TaqMan microRNA assays, which use target-specific stem-loop primers for cDNA synthesis, and SYBR Green dye-based chemistries with universal cDNA synthesis. These assays can offer high sensitivity and specificity and can be run on a range of q-RT-PCR instruments. Unfortunately, TaqMan assays cannot provide absolute quantification, which can lead to issues such as false positives and variability in results¹³. Also, despite their high sensitivity, the application of these assays in differing sample types can be limited by the need for extensive sample preparation and potential interference from other components in the sample.

Next-Generation Sequencing (NGS): miRNA-seq is increasingly popular for differential expression analysis across the entire miRNome of biofluids. A benefit of NGS technologies like miRNA-seq is that they allow for massive parallel analyses of the genome-wide expression of miRNAs, enabling the quantification of individual miRNAs, identification of sequence variations, and discovery of novel miRNAs¹⁴. A substantial drawback to sequencing-based methods, such as IlluminaTruSeq Small RNA Sample Preparation Kit, is the induction of quantitative biases, affecting the accuracy of differential expression analyses, due in large part to the homology and smaller read lengths of miRNAs¹⁴. Not only that, but the presence of isomiRs (variants that differ in sequence or length from the canonical miRNA sequence) and technical artifacts can complicate data interpretation, potentially leading to misdetections and confounding factors in biomarker assays¹⁴. Also, different tools for miRNA-seq data analysis, such as sRNAbench and miRDeep2, can yield varying results, affecting the consistency and reliability of novel miRNA discovery^15,16. Even something like microarray technology can be insufficient in most cases. Though it can provide a powerful, high-throughput platform for detecting miRNA or gene expression levels, making it suitable for genome-wide studies, the technology is still limited by the number of previously identified miRNAs and cannot directly verify miRNA-target interactions, which limits their utility in functional studies¹⁷.

The most important factor that can be stressed here when comparing all these available options is the lack of spatial context. They do not provide any context in relation to the tissue morphology or spatiotemporal distribution within the cells. Visualizing the spatiotemporal behavior of miRNAs is necessary to unravel their function at the cellular and subcellular levels. Presently, miRNAscope in situ Hybridization (ISH) assays are accessible, usually utilizing chromogenic detection methods. While these assays do allow for the visualization of miRNA with single-cell resolution, the results are lacking in quality, making it difficult to differentiate between the miRNA expression and the background of the sample. Generally, many of these produce low-quality assays, which are unreliable and usually require manual quantification.

Therefore, we developed MicroRNA Amplification and Recognition through Locked-nucleic-acid in situ hybridizatioN (MARLIN) using multiplex immunofluorescence staining (by Opal protein staining) and locked nucleic acid (LNA) probes targeted to a miRNA, within formalin-fixed paraffin-embedded (FFPE) tissues. We accomplished this by implementing a Tyramide Signal Amplification (TSA) system that is activated by an Anti-Digoxigenin (DIG) Horseradish Peroxidase (HRP) Conjugate antibody. The HRP acts as a catalyst to the TSA, while the Anti-DIG attaches to the LNA probe. This assay is of particular use in understanding the essential nature of miRs in post-transcriptomic modification with proteins, and potentially other RNAs simultaneously. Furthermore, due to the lack of a commercially available assay that can quantify both miRNA and protein expression, MARLIN provides an effective way to understand the in situ relationships of miRs and proteins within FFPE samples.

The initial results from the quantification of miR-181c-3p using MARLIN demonstrated the feasibility of parallel analysis of miRNAs and proteins within the native spatial context of human ovarian FFPE tissue. Beyond this example, based on the components of MARLIN, it is theoretically possible to expand to up to four proteins utilizing commercial fluorescent multiplexing fluorophores like Alexa Fluor, or other small molecule dyes¹⁸.

In addition, since this methodology employs variations on standard histology methods, it should be compatible with other mammalian FFPE tissues, given appropriate probe and antibody selection. This methodology preserves spatial architecture and allows the quantification of cellular and subcellular miRNA and protein colocalization. This approach addresses a critical gap in spatial biology by mapping miRNAs-protein co-expression patterns and spatial distribution, which can be directly contextualized within native tissue architectures. Here we present MARLIN with a detailed graphical overview in Figure 1. While the protocol presented is optimized for miR-181c-3p, other miRs are possible to study, given an appropriate probe design. Critical parameters for this method include: the assessment of tissue quality and the optimization of probe concentrations. It is recommended to assess RNA integrity of the tissue block using a DV200 metric by utilizing two 10 µm sections and extracting the RNA using a protocol that preserves all RNA fragment sizes. Samples with a DV200 score greater than 50 or better, and protocol concentrations, need to be systematically tested.

This protocol uses formalin-fixed paraffin-embedded (FFPE) tissues of human ovarian tumor tissue sections from patients with HGSC. These samples were collected from previously untreated patients undergoing primary cytoreductive surgery for ovarian cancer. All samples and clinical data were obtained from the ovarian cancer repository of the Department of Gynecologic Oncology and Reproductive Medicine under protocols approved by the University of Texas MD Anderson's Institutional Review Board. Written informed consent from the patients was obtained by front desk personnel, and the studies were conducted in accordance with recognized ethical guidelines.

1. Tissue preparation

1. Bake all FFPE samples overnight at 60 °C and clean each piece of equipment and tools with RNAse-free water. Ensure the sections are around 5 µm thick.
   NOTE: This protocol has been optimized for ovarian tumors but can potentially be used with other tissue types after further testing.
2. Perform deparaffinization with three rounds of Xylene for 5 min each round, and 10 rounds of rehydration for 3 min each round (100% ethanol (EtOH) 2x, 95% EtOH 2x, 70% EtOH 2x, 50% EtOH 2x, 30% EtOH 2x). Wash with 1x tris-buffered saline (TBS) for 2 min, once.
   NOTE: This dewaxing step, although recommended, is optional and dependent on tissue type.
3. After deparaffinization and rehydration, apply a solution of 3% Hydrogen Peroxide (H₂O₂) for 10 min. Wash for 2 min with 1x TBS, 2x. Then, apply a solution of 3.7% Paraformaldehyde (PFA) for 10 min. Wash for 2 min with 1x TBS, 2x.
   NOTE: This step is optional and should only be considered for fatty tissues, or samples that are prone to detachment.
4. Proceed to the first round of antigen retrieval. This protocol uses the EZ-Retriever IR System. Following the system's instructions, microwave the samples while submerged in fresh EZ-AR1 buffer at 95 °C for 15 min. Leave to cool for 20 min.
5. Wash for 2 min with 1x TBS. Then for 2 min with 1x TBS-T (1x TBS with 0.1% Tween), 2x.

2. Protein application

1. Draw a barrier around each tissue section with a hydrophobic barrier pen.
2. Add 1x Antibody blocking solution (enough to cover the entire tissue area) and incubate for 30 min at room temperature in a humidity chamber (ambient and uncontrolled humidity).
3. Subsequently, dilute the first primary antibody with the same blocking solution, apply enough to cover the entire tissue area, and leave to incubate for 1 h at room temperature in a humidity chamber.
   NOTE: During the development of this protocol, Anti-CD8 was used at a dilution of 1:75.
4. Wash for 2 min in 1x TBS-T, 3x. Then, add the Anti-Ms + Rb HRP and leave the samples to incubate for 10 min at room temperature in a humidity chamber (ambient and uncontrolled humidity).
5. Wash for 2 min in 1x TBS-T, 3x. Dilute the first fluorophore with the amplification dilutant at 1:100 and incubate for 10 min at room temperature in the humidity chamber.
   NOTE: During the development of this protocol, a 690 nm emission fluorophore was used. From this point, it is recommended to use minimal lighting for the rest of the procedure after applying the first round of fluorophore.
6. Wash for 2 min in 1x TBS-T, 3x. Begin the second round of antibody staining with the second round of antigen retrieval in the microwave, by again submerging the samples in fresh EZ-AR1 buffer at 95 °C for 15 min, then cooling for 20 min.
7. Wash for 2 min with 1x TBS. Then wash for 2 min with 1x TBS-T, 2x.
8. Apply the 1x Antibody blocking solution again for 30 min in a humidity chamber at room temperature (ambient and uncontrolled humidity).
9. Dilute the second primary antibody with the same blocking solution, apply enough to cover the entire tissue area, and leave to incubate overnight at 4 °C in a humidity chamber for at least 16 h.
   NOTE: During the development of this protocol, Anti-EpCAM was used at a dilution of 1:200.
10. Begin the following day by washing for 2 min in 1x TBS-T, 3x. Then add the Anti-Ms + Rb HRP and leave the samples to incubate for 10 min at room temperature in a humidity chamber (ambient and uncontrolled humidity).
11. Wash for 2 min in 1x TBS-T, 3x. Dilute the second fluorophore with the amplification dilutant at 1:100 and incubate for 10 min at room temperature in the humidity chamber.
    NOTE: During the development of this protocol, a 620 nm emission fluorophore was used.

3. Probe application

1. Wash for 2 min in 1x TBS-T, 3x.
2. Start the third and final antigen retrieval with EZ-AR1 Elegance in the microwave at 107 °C for 15 min. Leave to cool for 20 min. Wash for 3 min in 1x TBS.
3. Apply RNAscope Protease Plus (enough to cover the entire tissue area) and keep at 40 °C for 30 min. Use an oven. Wash for 5 min in 1x TBS, 2x.
4. Perform probe preparation by denaturation at 90 °C for 4 min of the miRNA LNA probe. Then immediately dilute the probe using miRNA-ISH buffer. Vortex and spin the probe solution.
   NOTE: During the development of this protocol miR-181c-3p target probe was used at a dilution of 1:250.
5. Apply probe (enough to cover the entire tissue area) and place back in the oven for 1 h at 60 °C. Wash for 5 min in 5x Saline Sodium Citrate (SSC) buffer.
6. Perform five stringent washes in SSC prewarmed at 60 °C in the following order and dilutions for 5 min each round: one round of 5x SSC, two rounds of 1x SSC, and two rounds of 0.2x SSC.
7. Wash for 5 min in 0.2x SSC at room temperature. Incubate with blocking buffer for 30 min at room temperature in a humidified chamber. Use a combination of 1x TBS and 0.5% Blocking Reagent Powder, make sure to vortex and spin the blocking solution before application.
8. Apply an Anti-Digoxigenin (Mouse) HRP Conjugate (for these samples, a dilution of 1:500 was used) and incubate for 30 min at room temperature in a humidified chamber.
9. Wash for 5 min in 1x TBS-T, 3x Apply a recommended dilution of 1:50 of the Tyramide Signal Amplification Plus Cyanine 3 (TSA+Cy3) fluorophore for 10 min in a humidity chamber at room temperature (ambient and uncontrolled humidity). Make sure to vortex and spin the TSA+Cy3 solution before application.
10. Wash for 5 min in 1x TBS-T, 3x.

4. Nuclear staining and mounting

1. Apply 4',6-diamidino-2-phenylindole (DAPI) working solution (enough to cover the entire tissue area) for 5 min at room temperature in a humidified chamber (ambient and uncontrolled humidity).
   NOTE: For this protocol, a dilution of 1:1000 was used, with the working DAPI stock diluted in TBS.)
2. Wash for 5 min in 1x TBS-T, 3x. At completion, apply coverslips using mounting media and leave slides to dry for 10 min. Store slides at 4 °C in the dark between 24-72 h before imaging and up to three months after.

5. Imaging and analysis

1. For imaging, use a slide scanning microscope (preferred) at 40x magnification.
2. Perform subsequent quantitative analysis to enumerate puncta utilizing DAPI and protein counterstaining for cell segmentation and phenotyping.
   NOTE: During this protocol, we utilized imaging with a Phenoimager HT. Full slides scans were captured at a 40x magnification utilizing appropriate filters for the fluorophores present, and multiplexed images were then exported as OME-TIFF files. The scanned images were then uploaded into Visiopharm Image Analysis Software for quantitative analysis.

When testing this protocol, it is important to have two serial sections of the same representative sample for accuracy and comparison. In this case, a High-Grade Ovarian Cancer (HGSC) sample was used to compare the results. A control slide (Figure 2A-B) that would only go through the miRNA staining and a test slide that would undergo both protein and miRNA staining (Figure 2C-J) were used.

Both the control slide and the test slide had consistent miRNA (miR-181c-3p) expression, with higher expression appearing in the tumor cells (Figure 2A-D). There also appeared to be minimal loss of miRNA signal between the two sections, indicating that the protein staining method used on the tissue was not overly harmful or damaging. This is a positive indication of the integrity and reliability of the staining method used in this protocol. As shown in Figure 1C-D, the application of the Anti-EpCAM antibody and the 620 nm emission fluorophore stained the HGSC ovarian tumor cells (Figure 2E-F). The application of the Anti-CD8 antibody and the 620 nm emission fluorophore (Figure 1B-C) marked the CD8+ T cells (Figure 2G-H). When combined, protein and miRNA detection provide a cell-type-specific spatial patterning of miR-181c-3p within the microenvironment of HGSC samples (Figure 2I-J).

As can be seen in Figure 3, the target probe needs to be a 5-prime LNA Detection Probe labeled with Digoxigenin (DIG), a steroid used for antigen detection. The Anti-Digoxigenin (Mouse) HRP Conjugate antibody used then recognizes the DIG and attaches. Horseradish Peroxidase (HRP) acts as an enzyme that catalyzes the reaction of hydrogen peroxide. The TSA is activated by HRP and then covalently binds to all TSA proximal to the HRP18. These tyramides, activated by peroxidases, will then either bind tyrosine amino acids or dimerize, which generates the background noise. Finally, the miRNA LNA probe is labeled by the TSA+Cy3 fluorophore, allowing for the visualization of both miRNA and proteins within the sample tissue section.

Optimization involved the use of a positive control (a U6 snRNA probe) and a negative control (a Scramble-miR probe), both at a dilution of 1:500. U6 is a small housekeeping RNA expressed in most tissues, making it an ideal positive control probe. While the Scramble-miR probe (negative control) did not share homology with any miRNA sequence, including the target, since the sequences were randomized. To validate the accuracy and reliability of the assay, tests were conducted on normal (non-cancerous) samples. There was no detectable signal expressed in the negative control of the HGSC sample along with the normal ovarian tissue and the normal fallopian tubes (Figure 4). The target probe was expressed only in the HGSC sample but not in the normal ovarian tissue, demonstrating the specificity and accuracy of the probe and assay.

Figure 1: MARLIN Protocol. (A) After baking the samples overnight at 60 °C, deparaffinization and rehydration are performed, and 3% H2O2 treatment for autofluorescence quenching. (B). Then there is the first round of antigen retrieval, blocking buffer, anti-Protein 1 antibody (in this instance, anti-CD8), HRP, and the 690 nm emission fluorophore. (C) A second round of antigen retrieval, blocking buffer, and anti-EpCAM antibody are used for hybridization. (D) The following day the HRP, 620 nm emission fluorophore, is added. The third round of antigen retrieval is performed, followed by protease plus incubation. (E) The miRNA probe is hybridized, followed by stringent washes, blocking buffer, and anti-DIG-HRP incubations. (F) The TSA+Cy3 is added, followed by DAPI incubation. Slides are then mounted with coverslips and scanned in a slide scanner. Once the slides have been scanned, image analysis software can be used to count puncta per cell and ploy glob analysis, as well as co-localization. This can be achieved by identifying individual cells through nuclear counter-staining-based cellular segmentation. Please click here to view a larger version of this figure.

Figure 2: Two serial sections of a representative HGSC sample stained with the multiplexed Protein and miRNA FISH. (A-B) miRNA detection protocol only. (C-J) MARLIN protocol utilizing both miRNA and protein detection protocol (EpCAM and CD8, and miR-181C-3p). Scale bars = 45 µm. Please click here to view a larger version of this figure.

Figure 3: miRNA probe reaction with the representative example of Anti-DIG-HRP, 3% H2O2, and TSA to provide signal and amplification. Please click here to view a larger version of this figure.

Figure 4: Results of the optimized MARLIN implemented on a representative HGSC sample, normal ovarian tissue, and normal fallopian tubes. (A, B) positive control U6 snRNA Probe expressed in the HGSC sample, (C) normal ovarian tissue, (D) and normal fallopian tubes. (E, F) The negative control Scramble-miR Probe HGSC sample, (G) normal ovarian tissue, (H) and normal fallopian tubes. (I, J) The target miR-181C-3p probe expressing in the HGSC sample, (K) and not expressing in normal ovarian tissue, (L) and normal fallopian tubes. Scale bars = 250 µm. Please click here to view a larger version of this figure.

Though the importance of miRNAs is recognized and understood, observations of both normal development and disease states are currently hindered by the difficulty of studying miRNAs using standard methods. The scarcity of in situ methods led to the development of MARLIN (miRNA FISH assay). Given the homology and short length of miRNAs, it becomes a challenge to visualize their location and effect on post-transcriptomic regulation. This protocol development utilizes Locked Nucleic Acid Probes (LNA). LNAs are modified RNA bases in which the ribose is locked with a methylene bridge, connecting the oxygen atom to the carbon atom. This additional bridge limits the flexibility normally associated with the ring, essentially locking the molecular structure into a rigid conformation¹⁹. LNAs enable a shorter probe and higher quenching efficiency, which supports assay design for lower background signal, and improved assay sensitivity and specificity.

Critical steps
The primary concern of this protocol design was probing specificity and sensitivity. To resolve this issue, this protocol utilizes Tyramide Signal Amplification (TSA), which is a technology that improves sensitivity by up to 100-fold while allowing the reduced consumption of probes. Additional optimization steps included the application of Protease Plus, which is gentle and controlled in its protein digestion, and can be advantageous for preserving tissue morphology and antigenicity in ISH-related protocols. The primary function of protease is to permeabilize tissue sections by partially digesting proteins, thereby exposing target RNA molecules (such as miRNA) so that probe hybridization can occur efficiently. The higher specific activity and thermal stability of this protease treatment are preferred where RNA integrity and exposure are necessary. If the protease step is applied first, it may destroy epitopes and weaken the protein signal. To address this, MARLIN utilizes protein immunofluorescence before protease treatment to preserve protein epitopes and the subsequent protocol optimization balanced miRNA accessibility with protein preservation. In addition, the inclusion of antigen retrieval techniques, which improve the sensitivity and specificity of detecting miRNA and proteins, can be carefully employed to reduce cell loss and better preservation of cell morphology, which is crucial for accurate analysis¹⁸. EZ-AR1 Elegance (pH 6) specifically was used during the final antigen retrieval to break the cross-links formed during formalin fixation of tissues and may contribute to a better signal-to-noise ratio in the assay, allowing for more precise quantification and localization of miRNA expression within the tissue samples.

The combined use of miRNA detection and protein capture assays allows for the simultaneous analysis of transcription and translation within the same cells, providing a comprehensive understanding of cellular functions and regulatory mechanisms. As previously mentioned, this is why in this protocol, protein staining was implemented first so as not to lose the integrity of the antigens when binding to the antibodies. During the two rounds of antibody staining, antigen retrieval EZ-AR1 (pH 6) was used for antigen retrieval solutions as it is compatible with multiplex immunofluorescence staining protocols and ISH. The fluorophores are organic dyes conjugated to antibodies and are used in a cyclic staining process where, after each staining round (primary antibody + fluorophore), the primary antibody complex is stripped off while maintaining the fluorophore in the original primary antibody location, and the next primary antibody is applied²⁰. This allows for the possibility of higher multiplexed staining without spectral overlapping due to sequential staining and spectral unmixing. In this protocol, three rounds of staining and stripping of primary antibodies did not appear to interfere with miRNA detection and quantification.

Limitations and modifications
There are three major potential drawbacks to this assay. The first is the limited panel size due to underlying limits on fluorophore combinations and imaging modalities. This possibly could be rectified by sequential immunofluorescence but requires further testing. The second is the need to design, test, and validate a custom miRNA probe. The third is that this is a manual, multi-day procedure. Nevertheless, these challenges are outweighed by the assay's ability to enable multiplexed detection of miRNA and protein, making it a valuable tool for targeted biomarker analysis. Though this protocol has been optimized specifically for ovarian FFPE tissue samples, other combinations of primary antibodies and miRNA are theoretically possible but would require optimization for dilution, epitope retrieval and probe design. Accordingly, dilutions of antibodies and miRNA probes will vary based on the sample and may require multiple rounds of testing. This is why the suggested dilutions in this protocol may not work for all tissue types. For the optimization of this protocol, anti-CD8 and anti-EpCAM antibodies were used to better identify stomal and tumor regions and morphology within the HGSC sample. The sequence for the miRNA probes should be oriented from 5' to 3' with preference for reducing GC, although a GC clamp is not mandatory. Prior to synthesizing the DIG LNA Detection Probe for the ISH, it is strongly recommended to evaluate the probe sequencing amplification capability using q-RT-PCR or other similar methods.

Comparison to other methods
MARLIN offers an alternative to combining two separate technologies-like multiplex immunofluorescence (Multiplex IF), which is a technique that enables simultaneous detection of multiple protein markers but cannot detect RNA, and miRNAscope-based methods, which provide qualitative results for miRNA expression. There are relatively few imaging spatial biology methods that can simultaneously detect miRNA and proteins. Furthermore, multiplex IF is also not always compatible with other assays, such as intracellular protein staining, limiting its utility in comprehensive cellular analyses¹⁸. As for in situ RNA methods, standard RNA probes usually target between 150-1,500 bps. RNAScope often has 20 regions pairs covering at least 300 bp. Even Basescope is known to cover 50 to 300 bps and does not consider the secondary structure or hairpin binding that often characterizes miRNAs. While miRNAscope is specifically designed to detect short RNAs between 17-50 nucleotides, small non-coding RNAs, siRNAs, ASOs, and miRNAs. However, miRNAscope lacks the quantitative elements for understanding the expression levels of different miRNA and instead is best applied as a binary call for the cellular expression of a miRNA²¹. Most other methods for measuring miRNA lack spatial context, such as hybrid methods similar to flow-FISH and sequencing-based protocols. While sequencing is untargeted, accurately mapping short reads is difficult when families of miRNA share similar homology. As a result, miRNAs are often overlooked during total RNA mapping and are instead typically studied using specialized miRomic kits. In contrast to sequencing-based methods, this manual protocol should be reproducible in most laboratories, processing multiple samples at a time, and still yield reliable results for analysis and spatial location for a targeted miRNA and protein set.

Conclusion
Here we have established and validated a small panel of two proteins with one miRNA detection. While it is possible to detect additional proteins using more fluorophores, scaling up this method remains a long-term goal for the future. This technique will still permit two additional fluorophores but will first require optimization to determine that serial retrieval and staining does not impact RNA integrity with each additional cycle. Results can be analyzed by puncta counts, or polyglob image analysis processing can be employed to quantify the miRNA expression in multiple tissue samples of various sizes and morphology. Future prospective applications would be to adapt MARLIN for automated systems, where complex automated protein sequential IF could possibly be integrated with miRNA detection.