Meeting the microRNA Profiling Challenge

Compared to other commercial microRNA profiling platforms, MiRXES’s ID3EAL qPCR platform stands out when used to discover and validate blood-based miRNA biomarkers.

From painful biopsies to invasive procedures, certain diagnostic exams have gotten a bad rap over the years for the discomfort they tend to cause. The 2008 discovery of circulating microRNAs (miRNAs) in human blood, however, promised to herald a new era in diagnostics. By simply analyzing the miRNA levels in patient blood samples or other biofluids, clinicians and scientists would be able to diagnose diseases with more ease and less pain on the patient’s end.

Still, quantifying miRNAs in biofluids remains a challenge: not only are they small, at around 22 nucleotides in length, but miRNAs also make up just 0.01 percent of total RNA in tissues. To make things even trickier, miRNA family members tend to have highly similar sequences. Hence, finding and distinguishing the different kinds of miRNAs is often easier said than done.

Various platforms for quantifying miRNA expression have since been developed through the years. However, their ability to quantify miRNAs in biofluids commonly used in diagnostics like serum and plasma remained unclear—at least, until now.

In a study published in Scientific Reports, researchers from MiRXES, Merck, National University of Singapore (NUS) and National University Hospital, Singapore (NUHS) put the different commercially available miRNA profiling platforms to the test. Here’s what they found.

Introducing the contenders

miRNA expression is typically profiled through one of three methods: reverse transcription quantitative polymerase chain reaction (RT-qPCR), next-generation sequencing and hybridization. As suggested by its name, in RT-qPCR, miRNA is reverse transcribed into complementary DNA (cDNA). In turn, the cDNA is amplified and detected through PCR—enabling the absolute quantification of miRNAs in a sample.

The specific and sensitive detection of miRNAs in RT-qPCR is made possible with the use of primers. Certain platforms, like that of Qiagen and Exiqon, use at least one universal primer that bind to adenosine tails attached to the ends of miRNA. However, the MiRXES ID3EAL qPCR assay relies on a proprietary three miRNA-specific primer approach—with each primer uniquely customized to a particular miRNA.

Meanwhile, miRNA sequencing (miRNA-Seq) reveals the sequence information for miRNAs present in a sample—providing a snapshot of the relative amounts of each miRNA in the process. But while the technique can rapidly identify novel miRNAs and distinguish different miRNAs down to the nucleotide level, the equipment required is much pricier compared to other techniques.

Finally, hybridization assays like the Nanostring nCounter involve the attachment of fluorescent barcodes onto the ends of miRNAs. A scanner then digitally counts the barcodes attached to the miRNAs of interest. Unlike RT-qPCR or miRNA-seq, hybridization requires neither cDNA conversion nor amplification—making it a simple, cost-effective technique.

The moment of truth

In the study, all the three profiling techniques were assessed including parameters like their reproducibility across different runs and ability to detect miRNA. Specifically, four different RT-qPCR platforms (MiRXES, Applied Biosystems, Qiagen, Exiqon) were evaluated alongside the NanoString nCounter and the Illumina NextSeq 500 system.

To assess the reproducibility of each platform, at least three independent runs on a reference serum were performed. With its three runs correlating to 0.99, our ID3EAL assay displayed almost perfect agreement—a figure matched only by miRNA-Seq.

Using a parameter called the lower limit of quantification (LLOQ)—the lowest number of miRNAs that could be qualitatively determined precisely by a platform—our platform also proved the most sensitive with a total of 438 miRNAs detected above LLOQ, edging out the likes of miRNA-Seq (372) and Exiqon (208). In contrast, the Nanostring platform detected the lowest number of miRNAs above LLOQ. This could be due to the lack of an amplification step, resulting in reduced specificity, combined with the low RNA input volume of 3 µL.

Interestingly, both our platform and miRNA-Seq managed to reliably detect the same 243 miRNAs, with this overlap indicating that the two techniques would complement each other well. Moreover, 246 miRNAs were solely detected by MiRXES, but not Exiqon or Applied Biosystem’s platforms. Thus, while RT-qPCR is already considered the gold standard in miRNA expression profiling, our technology still managed to stand out.

Two platforms, one goal

Ultimately, when it comes to detecting miRNA in reference serum, our ID3EAL qPCR assay comes out on top, with the highest sensitivity among all platforms. Despite the slightly lower detection rate of miRNA-Seq, its lower cost and faster turnaround time compared to qPCR-based platforms, coupled with its ability to detect novel miRNAs still make it an invaluable technique in the miRNA profiling toolbox.

Based on these results, future work on developing miRNA biomarkers could therefore proceed along a two-in-one approach: first, miRNA-Seq can be used to discover novel miRNAs, followed by targeted qPCR using the MiRXES ID3EAL platform for validation. By combining the strengths of the two techniques, researchers can comprehensively profile miRNA biomarkers and create innovative diagnostics in turn.