In addition to commonly studied messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), multiple types of RNAs have become evident over recent decades, including microRNA (miRNA), long non-protein coding RNA (lncRNA) and circular RNA (circRNA). However, the functions of these RNA types within our cells are not yet fully understood.
In this on-demand webinar, Dr. Boye Schnack Nielsen, manager of molecular histology at Bioneer, explores how, with the development of new in situ detection technologies, RNA in situ hybridization has now been enrolled in diagnostics applications, enabling the visualization of RNA localization in tissues and cells to better address their roles in the tissue microenvironment and in molecular pathways.
Read on for highlights from the live Q&A session or register to watch the webinar at a time that suits you.
BN: The TNF mRNA signal did not appear as dots because this was at the beginning, where I worked with the RNAscope to see whether this was a true RNAscope signal. After this, we should be able to see these dots. Since then, I have worked with many RNAscope probes and it is clear that in some cells, the abundance of the target sequence is quite high. Then the chromogenic stains have a very large area of homogenous rich stains where you cannot see individual dots. Particularly for those that are low abundant like the long non-coding RNAs, they appear often as single dots.
BN: Until now, we have not had a lot of experience with this miRNAscope assay from ACD. However, I think that both technologies may work. What I mentioned is that the sensitivity of the miRNAscope assay seems to be higher. I think this is what we should expect from this new assay. So, at least in case of the low-abundant miRNAs, I would go for the miRNAscope assay.
BN: We've worked with this AP version, and it is the red chromogen that fluoresces. So, we worked with that in the beginning and hoped that we can use the AP in fluorescence assays, but the problem was that it gives some bleed-through when we multiplex because there are two or three fluorescent filters on the slide. Then the AP red chromogen can give some difficulties with separation. I would use the HRP as this is what we use for our Ventana instruments that we use for rhodamine 6G substrate HRP assays.
BN: The stability of RNA is different. We have messenger RNAs that are these long pieces of RNA that are easily degraded. The circular RNA seems to be more stable in the way that they are not accessible for the usual RNAs to create them as miRNAs. Maybe you can detect circular RNAs in the wastewater. Maybe you can also detect some miRNAs, you can even detect miRNAs in feces, so why not also in wastewater? The reason why miRNAs could be stable enough is that they are probably protected by a capsule of proteins and that would be the reason or the possibility that you could detect them from wastewater.
Find out more about RNA in situ hybridization for diagnostics in this on-demand webinar>>