IDT simplifies miRNA discovery with miRCat launch
cloning libraries of small RNAs from any primary RNA source to
enable the discovery of new small RNA molecules.
The new kit, dubbed miRCat, contains all the reagents needed to clone small RNA molecules such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and endogenous small interfering RNAs (siRNAs) so that the resulting libraries can be sequenced and new small RNA molecules discovered. The system was first developed to aid in the discovery of new miRNA molecules which cannot be cloned using standard PCR (polymerase chain reaction) techniques as the molecules have a tendency to cyclise rather than clone, but is also suitable for cloning other small RNA molecules. To date there have been approximately 500 human miRNAs discovered and these have been associated with the regulation of approximately 30 per cent of all human genes. Distinct miRNA expression patterns have been associated with various types of cancer as well as playing a role in regulating cell development, metabolism and viral infections. There are various methods of visualising small RNA molecules such as Northern blots, qPCR (quantitative PCR) and microarrays but while these allow expression profiling they can only be used to detect known molecules. To identify new small RNA molecules a more complicated protocol is needed that involves amplifying the amount of RNA by cloning before identification of the molecules is conducted using sequencing techniques. "The single biggest problem with cloning miRNAs is that you have to put a cloning linker on these molecules to allow you to clone them and if you put a RNA ligase on these molecules, these molecules will cyclise between their 5' phosphate and 3' hydroxyl positions," said Dr Mark Behlke, vice president for Molecular Genetics at IDT. He explained that to overcome this problem the kit uses a pre-activated adenylated linking method developed by Lau and Bartel that was published in the journal Science. "Even though the Lau and Bartel method had been described in the literature it was not used as much as it could have been because the reagents were not commercially available and are not trivial to synthesise," said Dr Behlke. He explained that the RNA ligase enzyme needs energy from ATP (adenosine triphosphate to add a linker to the RNA, however this enzyme has a tendency to cyclise the miRNA rather than add the linker to the miRNA. To avoid this, the adenylation step is conducted in the absence of miRNA so that the enzyme can only adenylate the linker. As this is the high energy part of the reaction, the RNA ligase can then add the adenylated linker to the miRNA in the absence of ATP and avoid the cyclisation problem that would normally hinder the cloning step. "Two years ago, IDT started selling adenylated cloning linkers to help support these research projects, since then we have had lots of requests from end users for a kit that includes all the necessary cloning components along with optimised protocols," said Dr Behlke. According to the developer of the kit, Dr Rick Devor, Education Director at IDT, the cloning of the miRNAs was still a fairly complicated procedure and there were still a number of aspects of the procedure that required optimisation. These procedures are included in the kit along with all the oligonucleotides and adenylated linkers that required for the cloning procedure. "Even with the kit the cloning of miRNAs is not trivial and will take about a week to get to the sequences, the purification step is also not particularly easy but we managed to optimise the process so that it only takes an hour rather than half a day and that protocol is also included in the kit," said Dr Devor. He continued by stressing the importance of the technique for finding new regulatory RNA molecules that are being shown to play such an important part in disease onset and progression as well as possibly being useful as therapeutic candidates. "miRNAs are one of six known classes of small regulatory RNAs and are the only class that forms hairpins, this means that the number of possible miRNAs out there can be predicted on the basis conserved hairpins," said Dr Devor. "None of the other small regulatory RNAs form hairpins and so can't be predicted using algorithms - they can only be discovered by direct cloning and sequencing."
