NEW YORK (GenomeWeb) – Researchers at Thomas Jefferson University have developed a new method that can be used to quantify small non-coding RNA variants, including isoforms of microRNA known as isomiRs.
The method, called dumbbell PCR, involves using miRNA as a basis to create larger structures via ligation. These are then quantified with TaqMan RT-PCR. The technique provides single-nucleotide resolution at both the 5'- and 3'-terminal sequences, according to a proof-of-principle study published this week in Nucleic Acids Research.
The method gets its name because of the distinctive shape of the structure created. "The ligation product of the end loop adaptor and target RNA looks like a dumbbell," Yohei Kirino, a researcher at TJUs Computational Medicine Center and co-author of the study, told GenomeWeb in an interview.
MicroRNAs are about 20 nucleotides in length, but there can be many isoforms. "Some microRNAs are a little bit longer or shorter at either the 5' or 3' end, or both," Kirino said.
"It is very important to analyze single distinctive RNA species because one nucleotide can change the target RNA, and can change gene expression regulation," he said.
Until now, there has been no method that can easily accomplish analysis of the terminal nucleotide differences of RNA.
Kirino said researchers usually use Northern blot, PCR, or microarrays. "These three methods are based on the hybridization of primers to the target RNA," he explained, "But this hybridization cannot discriminate a target RNA from its terminal isoforms because the primer can bind both similarly."
Next-generation sequencing, meanwhile, does allow analysis of entire RNA species but it can be time-consuming and expensive, and requires subsequent bioinformatics. "It's not very easy to do next-generation sequencing only for single RNA expression analysis," Kirino said.
"Dumbbell PCR is useful because we use adaptor ligation to the target RNA terminals, [followed by] TaqMan RT-PCR, and the probe is targeted to the boundary of the target RNA and the adaptor," Kirino said. "By using Db-PCR we can amplify and quantify specific small RNA with specific terminal sequences."
The NAR study further demonstrated the method could be used on assorted endogenous small RNA species from various cell lines to identify different isomiRs.
Kirino's group initially wished to devise a novel method that could be easily performed, like PCR, to analyze specific variants of Piwi-interacting RNA and distinguish a new mutant form extended at the 3' end. But they found the method also worked quite well for microRNA and other small RNA species.
A study last month from another group at TJU suggested the number of microRNAs in humans might be much greater than previously estimated. Clinical applications of circulating microRNA analysis include evaluation of liquid biopsy biomarkers for cancer and neurodegenerative diseases, like Alzheimer's. It is possible that isomiR analysis may clarify some of the irreproducibility seen so far in this field. Isothermal amplification-based methods to analyze microRNAs are on the horizon, and ddPCR may also be an effective tool.
TJU is now pursuing a provisional patent on the method, Kirino said, but the researchers have no commercial collaborators as of yet.