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Tools & Techniques: microRNA Labeling, Production, and Quantification


NEW YORK (GenomeWeb) – Researchers from Vilnius University have developed a new method for the selective covalent labeling of microRNA and siRNA duplexes, which they claim can overcome challenges encountered with existing miRNA identification and profiling techniques.

Current methods for small RNA discovery rely on size-dependent cloning of nucleic acid strands followed by their massive parallel sequencing, the scientists wrote in a paper appearing in the Journal of the American Chemical Society. But these approaches lack the specificity needed to discern between molecules of interest and other types of RNA and DNA of similar size. Meantime, the use of in situ hybridization, DNA microarrays, and qPCR analyses is limited to known biological species.

To address this issue, the team combined a DNA and RNA labeling approach they previously developed called mTAG — short for methyltransferase-directed transfer of activated groups — with HEN1, a methyltransferase from Arabidopsis thaliana that transfers methyl groups from the co-factor S-adenosyl-L-methionine (AdoMet) onto the 2'-OH group of the 3'-terminal nucleotides of dsRNA molecules.

In their report, the research group demonstrated that the HEN1 methyltransferase can be used to attach desired functional and reporter groups at the 3'-ends of small RNA duplexes in either a two-step or one-step process.

This, it noted, is the first report of selective labeling of an entire class of cellular RNAs, and the method permits control in covalent manipulation of miRNAs and siRNAs, "paving the way to developing numerous novel techniques ranging from affinity purification, high-throughput profiling and specific single-molecule analysis using nanopore sensors."

To address the shortcomings of existing reagents for functional miRNA research, scientists from the University of California, Davis have created a system for the production of pre-miRNA molecules using Escherichia coli.

To date, the bulk of miRNA research involves expression systems based on recombinant DNA material, which tends to be inefficient because it relies on host cells or organisms to transcribe the DNA to miRNA precursors, or synthetic RNA molecules that consist of modifications that may alter an miRNA's natural biological activity, the group wrote in Drug Metabolism and Disposition. At the same time, in vitro transcription can result in RNA molecules of varying lengths.

Recently, however, there have been reports describing the use of tRNA and rRNA as scaffolds for the production of recombinant RNA in E. coli. Hypothesizing that this approach could be used to create RNA agents for functional research, the UC Davis investigators adapted it to produce active human miR-27b using the tRNA scaffold.

Recombinant tRNA/mir-27b chimeras were synthesized in the E. coli, then were purified to a high degree using anion-exchange fast protein liquid chromatography.

The tRNA-fusion mir-27b was processed into mature miRNA miR-27b in intestinal human colon adenocarcinoma cells in a dose- and time-dependent manner. Moreover, recombinant tRNA/miR-27b agents were biologically active in reducing the mRNA and protein expression levels of a known target gene, resulting in the expected phenotype.

"These findings demonstrate that pre-miRNA agents may be produced by recombinant RNA technology for functional studies," the researchers concluded.

To improve the reliability of stem-loop RT-PCR for miRNA quantification, a team of Hungarian scientists have refined the protocol for using the technology to account for the effects of total RNA input and DNA contamination.

Currently, quantifying miRNAs using RT-PCR involves cDNA synthesis, performed when a sequence specific stem-loop primer is hybridized to the mature miRNA and used to initiate the reverse transcription reaction, followed by real-time PCR, during which the extended and transcribed miRNA is quantified using oligos specific for the miRNA and the primer loop sequences.

In analyzing this method, the researchers discovered that increases in total RNA amount can result in lower apparent miRNA expression levels, the wrote in PLOS One. Further, they found that DNA could serve as a template during mature miRNA measurements, mostly during the reverse transcription reaction, affecting sequence detection.

Meanwhile, recently discovered isomiR species create a layer of sequence diversity that can be extensive on both the 5' and 3' ends. Their experiments, they wrote in their paper, indicate that 3' isomiR species of a particular miRNA can be reverse transcribed and cross-detected even by specifically targeted assays.

This problem, they added, could not be overcome even by using the poly(A)-tailing-based qRT-PCR methodology. "On the other hand, simultaneous reverse transcription of the target miRNA and the endogenous control does not necessarily influence the outcome of the results and may be a more accurate and cost-effective approach for miRNA level quantitation."

With these factors in mind, the investigators developed and described an improved protocol for the application of quantitative stem-loop RT-PCR for the accurate detection of mature miRNA species.