The discovery of miRNA (which resulted in the Nobel Prize in Physiology or Medicine in 2006
) was like a Big Bang in biology. Since the award, the number of articles describing the important roles of miRNA and RNAi has increased every year at the speed of light. To date, about 3,000 miRNA species have been discovered, regulating approximately one third of all protein-encoding genes important in development, differentiation, apoptosis, fat metabolism, viral infection, and cancer, to name a few. Your email account may be flooded every day with companies offering “the best”, “cheapest”, “most reliable” miRNA detection systems. So naturally, you may start thinking about incorporating miRNA detection into your research, but the question is, which methodology should you use.
Ambion (Applied Biosystems) has designed a very smart, reliable and easy to use detection system, enabling quantification of miRNA expression; the system is called the mirVana qRT-PCR Detection Kit. This detection system can be used to characterize expression of miRNA via both, classical endpoint RT-PCR as well as SYBR® Green-based real-time RT-PCR.
I use the mirVana qRT-PCR Detection Kit to detect various miRNA species in neurons along with pre-designed mirVana qRT-PCR Primer Sets, which can be ordered separately. An extensive list of primer sets, for almost every miRNA species, can be found on the Applied Biosystems website. This set up is very convenient. If you want to detect a new miRNA you can still use the left over reagents of the mirVana qRT-PCR Detection Kit from your previous miRNA detection. You just need to order a set of new primers.
As for any PCR reaction, you need to collect RNA first. We collect RNA for this analysis from a variety of samples: fresh brain tissue, cultured organotypic brain slices, cultured primary neurons, or single neurons, either freshly isolated or cultured. In any of these applications, the mirVana qRT-PCR Detection Kit works equally well.
Since miRNA species constitute only a small fraction of total RNA, detection of miRNA can be difficult. The mirVana qRT-PCR miRNA Detection Kit is very sensitive, and we were able to detect as little as 300 molecules of a particular miRNA species in a single neuron. Another important aspect of this kit is that it detects mature miRNA but not miRNA precursors, thus you detect only the functional form of miRNA. Also, each kit is conveniently supplied with controls: a miR-24 primer set and a total RNA isolated from human heart.
In summary, the mirVana qRT-PCR Detection Kit offers an unparalleled miRNA detection system, which can be strongly recommended to anyone interested in a precise determination of miRNA expression levels.