MicroRNAs (miRNAs) are a class of small, single-stranded, non-protein coding RNA molecules that regulate cellular messenger RNA and protein levels by binding to specific messenger RNAs. MicroRNAs play a crucial role in almost every aspect of cell biology, including developmental timing, proliferation, and apoptosis. Moreover, microRNAs are involved in various cellular activities, such as insulin secretion, immune response, neurotransmitter synthesis, and viral replication. Thus, aberrant microRNA expression has an impact on those critical processes, and consequently, leads to a number of pathological and malignant conditions.
It was observed that selective groups of miRNAs are commonly down-regulated or up-regulated in different types of human cancers and were frequently associated with cytogenetic abnormalities. For example consistent up-regulation of miR-17 and miR-21 was observed in prostate, colon, stomach, lung and pancreatic tumors and miR-155 was identified to be up-regulated in lung, breast and colon cancer. On the contrary, it was reported that miR-29 was down-regulated in chronic lymphocytic leukemia (CLL), acute myeloid leukemia, mantle cell lymphoma, breast, lung and liver cancer. Additionally miR-15a and miR16-1-3p was identified to be down-regulated in CLL, prostate and pituitary adenomas and let-7 family members were found to be down-regulated in breast, lung, colon, ovarian and stomach cancer.
According to the latest miRBase release (www.mirbase.org), more than 30,000 mature microRNA sequences are listed, with ~2500 human microRNAs identified up to date, which can target more than 30% of the human genome. Given the role miRNA plays in human diseases, recent studies have shown that microRNA expression profiles can serve for diagnostic tests on a molecular level for diseases, as well as bases for novel therapeutics.
Most importantly, recent identification of circulating microRNAs, have shown great potential of their use as biomarkers since they are readily available in blood samples. MicroRNA analysis, however, presents many challenges due to their low abundance, small size and sequence similarity between miRNA family members. Their small size makes their analysis more difficult compared to messenger RNAs, particularly with conventional molecular biology methods, such as polymerase chain reaction (PCR) and hybridization-based assays. The small size of probes that are used greatly affects the efficiency of these methods because of a very low melting temperature.
Since the development of massively parallel/next-generation sequencing (NGS) of nucleic acids, there has been an increase of microRNA identification and discovery. NGS involves preparation of a complemented DNA library from the RNA sample, followed by the sequencing of millions of individual molecules. Obtained sequence reads undergo a bioinformatical analysis to identify and quantify (relative abundance) both known and novel microRNAs using application tools such as miRDeep. Although NGS is a high-throughput assay for miRNA expression profiling, disadvantages of this technique includes the high cost, the high amount of RNA used, and the sequence-specific biases due to enzymatic steps in complemented DNA library preparation. Nonetheless, NGS has been recently used for differential expression analysis of miRNAs in different diseases, including ovarian cancer and Huntington’s disease, suggesting the usefulness of this technique for diagnostics and early detection.
It has been reported that microRNAs are found in body fluids, such as blood, saliva and urine, of both diseased and healthy people. These extracellular circulating microRNAs exist in a stable form and are resistant to endogenous RNAse activity, as well as extreme pHs and temperatures. The stability of circulating microRNAs are proven to be due to the fact that they are found to be packaged in the micro vesicles, such as micro particles and exosomes, or associated with RNA binding proteins, including Argonaute 2 (AGO2) and nucleophosmin (NPM1). Aberrant expression of circulating miRNAs in different diseases, such as stroke, cardiovascular diseases, breast cancer, ovarian cancer, gastric cancer, lung cancer, colorectal cancer, diabetes, hepatocellular carcinoma and drug induced liver injury has been reported. Blood-based biomarkers are attractive for cancer screening due to their minimal invasiveness, relatively low cost and ease of reproducibility.
MicroRNAs are also modified through a series of processing events after transcription like 5´-end phosphorylation, 3´- end adenylation or uridylation, terminal nucleotide deletion. The problem is that existing bioanalytical methods such as microarrays and a quantitative polymerase chain reaction are sensitive, but not capable of identifying the posttranscriptional modifications of microRNA. Thus, there is a need for a miRNA detection technique, which is direct and multiplexed, requiring minimal sample preparation and can provide qualitative information regarding these modifications.
In this publication, the research laboratory lead by Professor Maxim Berezovski at University of Ottawa reported a multiplexed microRNA detection technique based on capillary electrophoresis – electrospray ionization – mass spectrometry (CE-ESI-MS) that offers a convenient platform for label-free, direct analysis of miRNA from biological samples. CE is highly efficient, and versatile, CE separations are fast, relatively inexpensive, and robust, requiring small amounts of sample and reagents. Coupling CE with MS makes it a powerful method for analysis of biomolecules as it combines high-resolution separations with high detection selectivity and sensitivity.
Electrospray ionization is a soft ionization technique used in mass spectrometry for biomolecules. miRNAs can be directly observed without any amplification by mass spectrometry. On-line sample pre-concentration with desalting prior to CE-ESI-MS improves concentration sensitivity for detection of very low amounts of miRNA in complex biological samples without ionization suppression. Due to the size of mature miRNA molecules (21-23 nucleotides), the effect of nucleotide chain fragmentation can potentially be minimized. Therefore, CE-ESI-MS represents a promising method for endogenous miRNA expression and sequence analyses. The results for the CE-MS study were validated by conventional SYBR green-based quantitative reverse transcription PCR.
Using the CE-MS method, the researchers detected two endogenous human circulating microRNAs, a 23-nucleotide long 5´-phosporylated microRNA with 3´-uridylation (iso-miR-16-5p) and a 22-nucleotide long 5´-phosporylated microRNA (miR-21-5p) isolated from B-cell chronic lymphocytic leukemia serum. The CE separation and following MS analysis provides label-free quantitation and reveals modifications of microRNAs. MicroRNA profiling of serum samples with CE-MS has the potential to be a versatile and minimally invasive bioassay that could lead to better clinical diagnostics and disease treatment.
Citation: Khan N, Mironov G, Berezovski MV. Direct detection of endogenous MicroRNAs and their post-transcriptional modifications in cancer serum by capillary electrophoresis-mass spectrometry. Anal Bioanal Chem. 2016 Apr;408(11):2891-9.Anal Bioanal Chem