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Rockefeller Team Reports on New Method for microRNA Detection in Tissue Samples

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A research team led by investigators at Rockefeller University has developed a new method for detecting microRNAs in tissue samples, demonstrating its ability to differentiate skin cancer types based on tumor-specific miRNA signatures.

As miRNA research continues, more and more of these non-coding RNAs have been found to be specific to different tumor types, increasing interest in them as disease biomarkers. In line with this, a number of companies are actively developing miRNA-based cancer diagnostics, including Rosetta Genomics, which has five such products on the market.

However, there remains a need for more reliable quantitative laboratory tests to measure small, non-coding RNAs in archived, formalin-fixed paraffin-embedded, or FFPE, human samples with sufficient specificity and sensitivity.

Aiming to overcome this limitation, the Rockefeller scientists, led by RNAi pioneer and Alnylam Pharmaceuticals co-founder Thomas Tuschl, developed a multicolor fluorescence in situ hybridization, or FISH, protocol designed to enable the visualization of differentially expressed miRNAs in FFPE tissue.

The researchers described their method in last week’s Journal of Clinical Investigation.

“While miRNA microarray and/or real-time PCR analyses of fresh or archived materials can be used for molecular diagnostics, these approaches obliterate valuable cytoarchitectural details,” they wrote in JCI.

The team first worked to identify target miRNAs, extracting total RNA from 36 archived clinical materials and cultured cell lines from patients with basal cell carcinoma, or BCC, and Merkel cell carcinoma, or MCC, as well as from healthy individuals.

They ultimately identified two tumor-specific miRNAs: miR-205, which is associated with BCC, and miR-375, which is associated with MCC.

With these miRNAs in hand, they established their multicolor FISH protocol for use with FFPE tissue sections, revisiting RNA fixation, signal detection and amplification, and oligonucleotide probe design steps, according to the paper.

“Suboptimal RNA fixation leading to short and long RNA loss by diffusion, rather than RNA degradation, is the primary problem in optimizing RNA FISH,” they wrote.

To address this, they utilized a previously developed approach for overcoming the miRNA loss associated with conventional formaldehyde fixation that involves fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, or EDC, a water-soluble condensation reagent that “promotes phosphoamide bond formation between the miRNA 5’ phosphate end and aliphatic amines from amino acid side chains of surrounding proteins.”

Treatment with EDC was expected to prevent access of antibody-based signal amplification reagents to the target RNA-bound probe-conjugated haptens, and miRNA detection by directly labeled probes was not possible due to the small RNAs low abundance, according to the paper.

The scientists overcame this by systematically varying the linker length between the nucleic acid probe and hapten until they found a length that enabled antibody access and boosted signal amplification-based fluorescence detection.

“To amplify the hybridization signal of hapten-conjugated probes, we used tyramide signal amplification and enhanced the HRP-mediated oxidative tyramide coupling reaction by adding 4-bromophenylboronic acid,” the researchers wrote. “We confirmed optimal tyramide signal amplification for our reagent set by preparing Cy3-tyramide reagents and buffers for comparison with commercial Cy3-tyramide equivalents. After optimizing the reaction, we switched to tyramides of ATTO dyes that are brighter, more stable, and water soluble.”

During their experimentation, the team discovered mishybridization of locked nucleic acid-modified miRNA probes to ribosomal RNA through colocalization of signals to nucleoli, but were able to correct this through probe shortening and placement of LNAs outside of segments with rRNA complementarity.

With the optimized FISH RNA protocol and their target miRNAs in hand, the research team aimed to validate their approach in FFPE tissues sections from BCC and MCC patients.

“Amplified miR-205 and miR-375 signals were normalized against directly detectable reference rRNA signals,” they noted in JCI. Meanwhile, “tumors were classified using predefined cutoff values, and all were correctly identified in blinded analysis.”

Encouraged by the data, the researchers are working to develop their RNA FISH approach for molecular diagnostic applications and plan to next test it in larger sample collections, they wrote.

They added that although their current study focused on just two miRNAs in two types of skin cancer, the method is expected to be widely applicable since “EDC fixation pertains to all miRNAs in any FFPE tissue sample, probe design, and signal detection and amplification steps are flexible, and experimental conditions are constant.”

In a commentary accompanying the JCI report, Gennadi Glinsky, a Stanford University Medical School researcher and co-founder of Genlight Technology, said that the study provides “compelling experimental evidence” supporting the feasibility of multicolor RNA FISH technology in clinical laboratories for “highly accurate quantitative analyses” of miRNAs and other classes of disease-associated small, non-coding RNAs.