MicroRNAs are small non-coding RNAs 22 nucleotides in length. Their differential expression has made them attractive biomarkers for both normal and disease states. A common method for miRNA detection involves the extension of a complementary DNA probe with a reverse transcriptase. An alternative method, developed by New England Biolabs uses ligation. Specifically, two miRNA specific DNA probes are joined by an RNA splint and ligated using SplintR Ligase (NEB #M0375). The ligated product can then be amplified and detected by qPCR. This approach is sensitive and able to detect miRNAs that differ by a single nucleotide .
New England Biolabs provides a large selection of single-stranded RNA ligases. These enzymes are commonly used to join linkers to the ends of RNA for amplification and next gen sequencing. One unique enzyme is RtcB, it can ligate RNA with a 3’ phosphate to a 5’ OH acceptor. This is in contrast to the majority of RNA and DNA ligases that require a 5’ phosphate for ligation.
Small RNAs can be isolated by using the selective binding properties of the p19 protein. This 19 kDa protein tightly binds 19-21 bp dsRNA in a size dependent, sequence independent manner. The p19 protein can also bind longer RNAs if an RNA oligo, complementary to the 5´ end of the RNA, forms a 19-20 bp duplex. This method allows purification of endogenous RNAs to monitor their 5´ and internal modifications. The p19 magnetic beads can also be used for quantitative detection of RNA using labeled complementary probes. .
- Jin et al (2016), Sensitive and specific miRNA detection method using SplintR Ligase, Nucleic Acids Research 44(13):e116, doi: 10.1093/nar/gkw399
- Jin et al (2010) Protein mediated miRNA detection and siRNA enrichment using p19. Biotechniques. 2010 Jun;48(6):xvii-xxiii. doi: 10.2144/000113364.
- Wulf, M.G., Maguire, S., Humbert, P., Dai, N., Bei, Y., Nichols, N.M., Correa, I.R., Jr., Guan, S 2019. Non templated addition and template switching by Moloney murine leukemia virus MMLV based reverse transcriptases co occur and compete with each other J Biol Chem. 294(48), PubMedID: 31640989, DOI: 10.1074/jbc.RA119.010676
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How can more efficient ligation of RNA-splinted ssDNA fragments improve your ligation-based detection assay? Learn about PBCV-1 DNA Ligase, also known as SplintR® Ligase.