miRNAs have emerged as important fine-tune regulators of a large host of genes that play a role in many cellular processes and signaling pathways.
These molecules function in a variety of diseases, such as cancer and inflammation, highlighting their potential as therapeutics and diagnostic biomarker tools.
CHI’s upcoming “microRNA: Targets and Tools for Therapeutic Development” conference will address the latest discoveries regarding miRNA-mediated disease regulation and introduce new methodologies for conducting these studies in a laboratory or clinical setting.
Much of the latest research in miRNA profiling for biomarker analysis has focused on improving methods of miRNA detection. Daniel Pregibon, Ph.D., CTO of Firefly BioWorks, will discuss the firm’s latest technology, FirePlex™, a method of miRNA detection that has been used by both academic and clinical researchers.
Dr. Pregibon explains that there are two main classes of technologies widely available for miRNA profiling studies, either using microarrays or deep sequencing to profile all miRNAs in a single sample, or using single assays to examine individual miRNA expression across many samples. Unfortunately, these approaches are not suitable for performing biomarker validation studies, which are fundamental in the development of diagnostic tests.
“What we’re seeing is that there was really nothing in that middle range, if you wanted to look at 25–50 miRNAs over hundreds or thousands of different samples,” he says. Thus, FirePlex was designed to help researchers with high-throughput validation of a certain subset of miRNAs of particular interest for their studies.
In fact, because each researcher may be interested in looking at a unique set of miRNAs, Dr. Pregibon reported that 99% of the panels they have made are custom panels, which can be quickly prepared within one week. Additionally, FirePlex panels have been developed to run on benchtop flow cytometers, so additional equipment does not need to be purchased to run an assay.
The FirePlex technology is built on encoded hydrogel particles, instead of using a glass surface, as would be utilized in microarrays. Dr. Pregibon explains that using a hydrogel instead of a glass surface allows each molecule to have many more degrees of freedom, which is beneficial from a thermodynamics standpoint. This hydrogel base for capturing miRNAs allows for better sensitivity and specificity. Polyethylene glycol (PEG) is used as a substrate to eliminate nonspecific binding of proteins, lipids, or other complexes.
FirePlex can also be used with crude samples because the assay is based on post-hybridization labeling (the miRNAs are labeled after being hybridized throughout the hydrogel volume), so any debris will be washed away after capturing the miRNAs. Dr. Pregibon notes that minimal manipulation of the sample also reduces any bias that is introduced as a result of the RNA isolation method used, which can lead to variable RNA yield or selective enrichment. FirePlex has also been expanded to profile miRNAs in cell lysates, fresh tissue, FFPE, and serum/plasma.
SomaGenics has developed a method, miR-IDirect, for direct detection of miRNAs from a plasma sample without requiring total RNA isolation. Sumedha Jayasena, Ph.D., vp of technology and therapeutic development, says the miR-IDirect platform, which detects circulating miRNA in plasma, incorporates SomaGenics’ qPCR-based miR-ID technology.
The miR-ID method provides quantification of small RNAs by first circularizing them, then generating cDNA by rolling circle amplification of the miRNA circles, and finally further amplifying the cDNA by qPCR using 5´-overlapping PCR primers. miR-ID has previously been applied to detecting miRNAs in total cellular RNA as well as purified from various biological fluids.
Validated miR-ID assays have been developed for about 100 different miRNAs to date, with more in development. The miR-ID technology can discriminate miRNAs that carry terminal modifications, such as 2´-OMe groups at 3´-ends, according to Dr. Jayasena.
He says that the miR-IDirect technology was developed to overcome certain limitations that have been encountered in analyzing circulating miRNAs as potential biomarkers. He explains that the latest wave of miRNA-based biomarker research has been primarily focused on their identification in biological fluids, especially in blood, as it was discovered that miRNAs are surprisingly stable in biofluids, and their expression profiles often correlate with particular disease states.
Current methods require total RNA or miRNA isolation, which can lead to bias against certain miRNAs and insufficient sensitivity to detect those miRNAs that are present at low levels. Further, a normalization standard, based on an endogenous miRNA, has not been established for profiling circulating miRNAs. A control miRNA can be spiked into samples, but because (unlike endogenous miRNAs) it is unprotected from attack by plasma nucleases, it is likely to be degraded to an unpredictable extent.
miR-IDirect addresses these issues, providing more accurate miRNA detection and quantification. The method “should facilitate research on circulating miRNAs, and, with minor adaptations, miRNAs in other biological fluids besides blood,” Dr. Jayasena says. “Our technology will help eliminate variability associated with current methods, while improving sensitivity in miRNA quantification and accelerate clinical validation,” Dr. Jayasena states.