An explosion in interest in RNA-based tools for drug discovery has taken place in the last couple of years, and researchers are still working out the best ways to work with and analyse them.
A new technical note from RNA specialist company Ambion describes a technique that allows easier monitoring of short RNA sequences in a variety of sample types and is 100-500 more sensitive than Northern blot analysis.
The interest surrounding RNA has split into two different but inter-related areas areas. In one, small double-stranded RNAs (dsRNAs), also known as small interfering RNAs (siRNAs), have been used to silence the expression of specific genes at the post transcriptional level by a pathway known as RNA interference (RNAi). In the other, numerous small regulatory RNA molecules, referred to as microRNAs (miRNAs), have been shown to regulate target gene expression in various organisms.
"It is now becoming apparent that siRNAs and miRNAs are related molecules, sharing common processing pathways and maybe even functional mechanisms," notes Ambion.
Currently, most miRNA researchers are analysing miRNA expression patterns by Northern blot, a technique that is relatively insensitive and labour-intensive. A few researchers performing gene silencing experiments also use this technique to analyse siRNA levels after RNAi induction, although Ambion believes the majority of researchers performing gene silencing experiments do not monitor siRNA levels at all.
"This may be due in large part to the inherent difficulties in detecting small RNAs with standard techniques," says the company.
Ambion has developed a technique which relies on a simple principle. This is that hybridisation - a technique in which single stranded nucleic acids are allowed to interact so that complexes or hybrids, are formed by probes with a complementary sequence - is more sensitive when it is carried out in solution than when it is done on a solid support, as in Northern analysis.
The method uses the RNase protection assay, in which a radiolabelled probe is added to a sample, hybridisation is allowed to take place and excess unhybridised RNA and the probe is removed using the RNase enzyme. A reagent is then added that precipitates the nucleic acid and disables the RNase.
Ambion used the technique to test decreasing sizes of siRNA sequences and demonstrated that it was able to detect concentrations of as little as 50 attomoles (10-18 mol) of the target RNA, with good specificity. Only the corresponding RNA strands were detected, and no signal was generated if none of the target RNA was present. Moroever, the technique was able to detect a specified strand in a complex mixture of siRNA.
Gene expression studies
Similarly, the technique can be used to monitor the expression of two different miRNAs across mouse tissues. The function of most miRNAs is not known, notes Ambion, but a number of them seem to be involved in post-transcriptional gene regulation. For example, some (e.g. lin-4 and let-7) inhibit protein synthesis by binding to messenger RNA (mRNA) molecules, from which proteins are expressed.
Interestingly, the pattern of expression uncovered using the RNase protection assay was similar for one miRNA (miR-16) to that achieved with Northern blot, but completely different for another sequence (miR-22). miR-22 was highly expressed in lung and ovary and was present in spleen, thymus, and testicle at levels that would not have been detectable with standard Northern blotting techniques.
Ambion's technical note also demonstrates that the technique can be used to detect small RNA's and mRNA in a single sample, as well as to simultaneously detect siRNA expression and target gene knockdown.
"The experiments presented here indicate that the solution hybridisation assay based on ribonuclease protection can sensitively and specifically detect small RNAs such as miRNAs and siRNAs," said Ambion.