miRNA Tools Lead Development of Next-Generation Therapeutics and Diagnostics

miRNA Tools Lead Development of Next-Generation Therapeutics and Diagnostics

Although microRNAs (miRNAs) represent only a small fraction of the total RNA mass in a given sample - estimated at about 0.01% - the short RNA molecules (16-35 nucleotides) are widely viewed as a rich analytical resource for biomarkers and drug targets. miRNAs are believed to be involved in the regulation of more than 30% of protein-coding human genes through a complex regulatory network. Through broad gene regulation activity, miRNAs influence a range of cell processes and consequently play roles in various diseases including cancer, neurodegenerative disorders, and cardiovascular and autoimmune diseases. With potential applications in disease-specific diagnostics and therapeutics, miRNAs have become a focal point in the life science research tools market (instruments, reagents, procedures) profiled by Kalorama Information in MicroRNA Tools and Services Market. Tool markets in the report include qRT-PCR, hybridization (arrays) and next-generation sequencing (NGS) as well as miRNA research-specific reagents such as miRNA mimics and inhibitors.

Although microRNAs (miRNAs) represent only a small fraction of the total RNA mass in a given sample - estimated at about 0.01% - the short RNA molecules (16-35 nucleotides) are widely viewed as a rich analytical resource for biomarkers and drug targets.

miRNAs have been detected in many types of tissues, as well as extracellularly in biofluids such as serum, plasma, saliva and urine. As their expression can be downregulated or upregulated in various diseases and cellular states, the differential expression of miRNAs can serve as a non-invasive, cost-effective biomarker for the early diagnosis of various diseases, as well as for the prognosis of disease recurrence and therapy outcome.

Various characteristics of miRNAs, such as their biological stability under stringent conditions, render them particularly suitable as biomarkers for clinical applications. As opposed to mRNA, which is highly fragmented in formalin-fixed paraffin-embedded (FFPE) tissue, miRNAs are stable and intact even in FFPE, and are therefore a preferred analytic measurement in clinical and research settings. In contrast to the well-preserved miRNA, the RNA extracted from FFPE is usually of poor quality for molecular analysis, due to its degradation and chemical modification during formalin fixation, along with the enzymatic fragmentation that occurs during long periods of storage.

Various characteristics of miRNAs, such as their biological stability under stringent conditions, render them particularly suitable as biomarkers for clinical applications.

As the alteration of miRNA expression levels contribute to the development of disease, current miRNA-based therapies in development aim to correct the imbalance associated with different diseases. Thus far, scientists have employed two main approaches to address this imbalance, either by mimicking or inhibiting the function of particular miRNAs.

Currently, three major technologies are employed for profiling the expression of miRNA in various biological samples: quantitative reverse-transcriptase PCR (qRT-PCR), hybridization arrays, and next-generation sequencing. Techniques using these platforms were adapted mostly from messenger RNA (mRNA) or gene expression profiling techniques, though their optimal use with miRNA is only possible through specific experimental techniques and reagents discussed in detail in MicroRNA Tools and Services Market.

qRT-PCR is a well-established method that is considered the gold standard for the quantification of miRNA expression, as it is highly sensitive, reliable, and reproducible. The technique is widely used, easy to incorporate into laboratory workflow, and rather inexpensive, although the initial setup of PCR equipment in a laboratory might constitute a larger expense. The technique is also highly flexible in incorporating new assays for novel miRNA, and the qRT-PCR data does not require bioinformatics manipulation. qRT-PCR methods are more accurate, sensitive, and rapid, and demonstrated higher detection rate than microarrays and NGS.

Typically, qRT-PCR requires low sample input and is particularly useful when the sample is limited. Moreover, qRT-PCR is applicable to many different types of samples, including cells, fresh tissue, and FFPE.

qRT-PCR is a well-established method that is considered the gold standard for the quantification of miRNA expression, as it is highly sensitive, reliable, and reproducible.

The detection and profiling of miRNAs by qRT-PCR poses particular challenges, related to the short length of miRNA that necessitates special methods and primers. The low representation of miRNA in total sample RNA also necessitates costly and time-consuming extraction and purification. Amplification errors can also arise from reverse transcription, varying primer efficiency, and preferential ligation and amplification. Primer design for miRNA analytes must be highly specific to adequately discriminate between miRNAs in the same family that can differ by only a single nucleotide. Stem-loop primers exhibit increased specificity and sensitivity compared to linear reverse transcription primers, but the technique is more time-consuming and of lower throughput, as it requires the design of specific primers for each miRNA. Unlike sequencing, qRT-PCR is not an “open-read” technology for discovery and requires primers designed from previous knowledge of desired miRNAs.

Similar to qRT-PCR, hybridization arrays require prior knowledge of the target miRNA sequence and probe design is complicated by the short length and high specificity of miRNA structure. Nonetheless, microarrays offer many advantages for miRNA expression profiling as they are reliable, easy to use, rapid, cost-effective and capable of profiling thousands of miRNAs simultaneously in a single experiment. Microarrays are less expensive than qRT-PCR or NGS, and their high throughput capabilities rendered this technique very useful in miRNA research, particularly in large scale studies such as cancer biomarker validation.

Several analytical limitations, however, have limited the continued use of hybridization arrays in the high-growth market of miRNA tools. Microarrays have lower sensitivity and specificity than qRT-PCR and NGS techniques, mainly due to variations in the hybridization efficiency of probes and non-specific, cross-hybridization of miRNAs. As the simultaneous processing of a large number of samples on microarrays imposes the use of universal conditions for hybridization, the optimal conditions for individual probes may be affected, impairing their efficiency. Given the similarities in the structure of some miRNAs, non-specific hybridization can occur, leading to false positives; conversely, low, below-the-threshold, hybridization signals due to probe inefficiency can result in false negatives. In addition, microarrays have a lower dynamic range than qRT-PCR and NGS - typically about 4 orders of magnitude - and can suffer from high background signal and saturation effects. Microarrays measure only the relative abundance of miRNAs, and the quantification of low-abundance miRNAs is often difficult due to low signal-to-noise ratios.

Microarrays are less expensive than qRT-PCR or NGS, and their high throughput capabilities rendered this technique very useful in miRNA research, particularly in large scale studies such as cancer biomarker validation.

The sequencing of miRNA is accomplished using largely the same protocols as conventional RNA sequencing. The main differences lie in the sample preparation protocol, which does not require the fragmentation of miRNAs, given their naturally smaller length. However, the preparation of samples in this case imposes the use of particular strategies to reduce the formation of adapter dimers, and necessitates a final library purification step. Additionally, the RNA isolation and purification reagents and protocols need to be adapted such as to retain small RNAs and to preserve their structure.

Compared to qRT-PCR and hybridization, NGS has several advantages, including higher throughput and sensitivity, greater accuracy, broader dynamic range of more than 5 orders of magnitude, enhanced reproducibility, and ability to discover novel molecules. Additionally, NGS can provide both qualitative and quantitative assessments of miRNAs expression in one experiment. Also, sequencing crucially permits profiling of both known miRNA molecules and new, previously unknown molecules. Since NGS is independent of pre-designed probes, it is not limited by array content or primer design, allowing researchers to identify novel miRNAs that could not be detected by previous techniques.

Also, sequencing crucially permits profiling of both known miRNA molecules and new, previously unknown molecules. Since NGS is independent of pre-designed probes, it is not limited by array content or primer design, allowing researchers to identify novel miRNAs that could not be detected by previous techniques.

Moreover, sequencing permits the detection of miRNA molecules over a broader range of expression levels than microarrays and qRT-PCR, since miRNA’s natural expression levels range anywhere between a few molecules to tens of thousands per cell. NGS’s large dynamic range and the ability to detect unknown molecules render the technology very suitable for global miRNA profiling studies. NGS is also better at detecting subtle changes in expression and has a greater accuracy in distinguishing between highly similar miRNA sequences, differing by only one nucleotide, between isomiRs of different lengths, and between mature miRNA and its precursors.

Despite its advantages, NGS has had several limitations that have hindered its applicability to miRNA research thus far; these include its extensive sample preparation requirements, inaccuracies in quantitation, higher costs, greater complexity, and fragmented workflow requiring instrumentation, reagents and data analysis software from various vendors.