I recently came across a booklet regarding SmartFlare for live cell RNA level detection. The basic idea, if i understand it correctly, is the use of gold nanoparticles that are conjugated to fluorescently tagged short double stranded probes. The “capture” strand is bound to the gold particle, is non-fluorescent and complementary to the target mRNA. The “reporter” strand is complementary to the capture strand but shorter. It is fluorescently labeled, but the gold is quenching the fluorescence.
This particle can enter the cells and if the target RNA is present, it replaces the “reporter” strand which is set free to the cytoplasm and fluoresce. This fluorescent can then be detected by microscopy or any other fluorescent detection.
I haven’t used it yet because it clearly isn’t suited for single-molecule analysis. Furthermore, is does not give any data on RNA localization since the fluorescence appears only after the target RNA is bound and the “reporter” is a short oligo which probably diffuses very fast.
Furthermore, I’m concerned about the biological effects. Although they claim in their FAQ section that at the concentrations used it does not cause RNA knockdown and is non-toxic, I am concerned first about the short reported oligo which presumably will hybridize with antisense to the target RNA, if present and second, that capturing the target RNA will affect its gene expression (probably prevent its translation, maybe mis-localize it and can promote its decay thus actually affect its transcription rate as well).
Particularly I’m concerned due to the long incubation time they suggest in their protocol – 18 hours. Why so long? Shouldn’t it work at shorter time-scales?
This system can be a good reporter for relative expression levels for whole cell populations or single-cell analysis, but I’m hesitant about the use of these same cells for downstream assays.
Also, their illustrations always show complementary of the capture strand to the 5′ end of the target RNA. Does it have to be the 5′ end?
I am considering the use of this system as a reporter for my experimental purposes. Any thought? Did anyone use that and share their experience?