From bench to breakthrough: Streamlining miRNA research

MicroRNAs (miRNAs) are small but mighty. Even the 2024 Nobel Prize in Physiology or Medicine acknowledged their significance.??

These powerful regulators of gene expression serve as precise diagnostic and prognostic biomarkers for diseases like cancer and neurological disorders. However, their small size and unique properties require specialized workflows for accurate analysis.?

This month’s newsletter outlines a comprehensive miRNA research workflow, from high-quality extraction to biomarker discovery with next-generation sequencing (NGS) and precise quantification using digital PCR (dPCR).?

Step 1: High-quality miRNA isolation? ?

Most RNA isolation kits focus on mRNA, but specialized kits are essential to capture small RNA fractions like miRNAs. Advances in silica-based spin column methods have improved RNA yield and purity while reducing contamination. The choice of sample type (e.g., cell cultures, tissues, blood) is also critical for success.?

Quick tips for isolation success:?

1. Stabilize RNA immediately after collection using reagents like RNAprotect or Allprotect.?
2. Optimize disruption methods with tools like the TissueLyser for tough-to-lyse samples.?
3. Choose phenol-free kits like miRNeasy Advanced to minimize gDNA contamination and capture miRNAs efficiently.?

For more detailed tips, check out our technical guidelines on miRNA isolation.?

Step 2: Discovery with NGS? ?

NGS enables comprehensive profiling of thousands of miRNAs, identifying known and unknown sequences, isoforms and expression levels across diverse samples. Advanced library preparation, such as QIAseq miRNA Library Kits with Unique Molecular Indexes (UMIs), addresses challenges in miRNA sequencing like amplification bias and contamination. Gel-free workflows and automation further enhance reproducibility.?

Accurately measuring miRNA abundance and expression profiles is critical for:?

• Identifying specific disease states or therapeutic responses?

• Understanding pathways for targeted interventions?

• Deciphering regulatory networks and molecular mechanisms?

Quick tips for NGS success:?

1. For biofluid samples, consider enriching for exosomes to target miRNAs that are better preserved.?

1. Eliminate small RNA contaminants, such as Y RNAs, during library preparation to capture target miRNAs efficiently and use spike-in controls to monitor workflow efficiency.?

1. Plan for a sequencing depth of 10–20 million reads per sample to ensure comprehensive coverage and sensitivity and use UMI-based analysis to avoid over- or under-sequencing.??

Watch our webinar to explore how NGS reveals novel miRNA expression patterns.?

Step 3: Verification with digital PCR? ?

Verification translates discoveries into actionable biomarkers. dPCR, unlike qPCR, directly counts target molecules for precise, reproducible results, even in challenging samples. The QIAcuity system quantifies miRNAs with as few as 5 copies per reaction, while miRCURY LNA PCR Assays improve specificity.?

Quick tips for dPCR success:?

1. Use LNA-enhanced primer sets to improve mismatch discrimination and for single-nucleotide differentiation.?
2. Start with appropriate RNA inputs for the RT reaction (1 pg to 100 ng of total RNA) for consistent results across different input levels without pre-amplification.?
3. Include spike-in controls during reverse transcription and amplification for accurate normalization, especially when working with biofluids or low-input samples.?

Download our poster on miRNA quantification with dPCR.?

Step 4: Data analysis: Integrating discovery and verification? ?

Successful miRNA biomarker research requires structured data analysis, from exploratory sequencing to precise validation.?

Quick tips for data analysis success:?

1. Utilize advanced visualization tools, such as the RNA-seq Analysis Portal (RAP), to interpret data comprehensively through volcano plots, heatmaps and statistical outputs.?
2. Incorporate Ingenuity Pathway Analysis (IPA) to connect miRNAs to biological pathways and disease mechanisms and prioritize biomarkers based on functional relevance.?
3. Integrate miRNA expression data with functional assays for further sample classification, molecular mechanism investigation and gene expression correlations, improving biomarker validation.?

Learn more about mastering data analysis using RAP and IPA.?

Spotlight on some innovative research?

Researchers at the Texas Heart Institute are investigating differential miRNA expression in heart disease from a biorepository of cryopreserved biofluid samples. Here’s what they had to say about getting to the heart of miRNA profiling.?

Meanwhile, Hummingbird Diagnostics is developing a blood-based screening test for lung cancer, focusing on small RNAs (including miRNAs) to capture the host immune response. In this article, you will read how their pipeline combines NGS discovery with dPCR verification to improve test performance.?

Explore the miRNA resource center for informative webinars, tools and services to support your research.??

What tool are you missing? Leave us a comment below!?