What standard methods are currently used to measure microRNA (miRNA) biomarkers?

A: Typically, miRNA biomarkers are measured with quantitative polymerase chain reaction (qPCR) after the RNA is converted to complementary DNA (cDNA). Since miRNAs are only about 20-25 nucleotides in length, though, before they are converted to cDNA a longer molecule is generated that will work as a qPCR substrate. Valid strategies for accomplishing this include poly-adenylation, adapter ligation, and application of miRNA-specific stem-loop reverse transcription primers. Our group and many others rely on the latter method, which was described in the scientific literature more than 10 years ago (Nucleic Acids Res 2005;33:e179). Why? Stem-loop qPCR efficiently discriminates between the unprocessed precursor miRNA and mature miRNA, uses a fluorescently labeled hydrolysis probe that imparts additional specificity, and achieves better technical reproducibility compared with other systems we have tested.

What challenges do clinical laboratories face when using miRNA biomarkers?

miRNA-based diagnostics are finally entering the clinic. To give an example, qPCR miRNA assays are now available to help endocrine surgeons better classify thyroid cancers. However, these kinds of tests are likely best suited to a companion role. In contrast with DNA or RNA-based tests that indicate the presence of a mutation(s), miRNA tests produce results that are difficult to interpret. Most miRNAs are expressed widely in a non-cell-specific manner, and they do not differ drastically in level between cases and controls. In fact, a truly disease-specific miRNA probably does not exist, whether in cancers or in non-neoplastic diseases. Many miRNAs proposed as biomarkers for one disease have been found in association with a bewildering variety of other conditions (PLoS One 2014;9:e89565).

Although very stable, miRNAs also contain little information. As a result, successful miRNA-based tests must often measure ten or more miRNA species with varying weights assigned to each. In particular, liquid biopsies in which blood, urine, or other fluids are gathered present special difficulties compared with tissue sampling, as miRNA levels are very sensitive to pre-processing and post-processing factors.

Data processing steps come with challenges as well, including normalization or adjustment to invariant miRNAs or other analytes. When selecting tools for miRNA test data analysis, clinical labs should ensure strict standardization of the entire process, from obtaining and processing the sample through results reporting. This is the key to reproducible and potentially informative miRNA results, no matter what technology is used.

Could emerging assays facilitate clinical labs’ adoption of miRNA testing?

Recent innovations in miRNA detection include amplification-free technologies, assays that do not require extensive RNA purification, detection of signal by flow cytometer, and departure from traditional cDNA generation, such as using the miRNA itself as a primer. These developments may simplify adoption of miRNA testing in clinical laboratories. An established technology, digital PCR (dPCR), also provides a quasi-absolute readout or copy number for miRNAs, at least for assays that have been thoroughly validated. dPCR thus eliminates the need for standard curves as well as the influence of normalization strategies. However, even with these advances, the other challenges of miRNA biomarkers still remain.

Kenneth Witwer, PhD, is an associate professor of molecular and comparative pathobiology at the Johns Hopkins University School of Medicine in Baltimore.+Email: [email protected]