Interphase 4: What about formaldehyde?
A recurring question is if formaldehyde (FA) crosslinking can be used for XRNAX. So far, we have not used FA-crosslinking for XRNAX. While we do not exclude the possibility that FA-crosslinking could be used, we do see a number of conceptual problems ahead. These issues arise from the fundamental differences between UV-crosslinking and formaldehyde crosslinking and therefore run diametral to the favorable features of UV-crosslinking described in the previous post (Interphase 3).
First, FA crosslinks everything. That means indirect contacts between protein and RNA via other proteins or DNA can be formed. So even if the extraction works out, one has to ask the question, what interactions are we looking at?
Second, FA crosslinks everything. That means it is expected that DNA in the interphase will be crosslinked to protein as well. A very central feature of XRNAX is that it eliminates DNA. Evidently, DNA cannot be digested to completeness when FA-crosslinked. Therefore, one has to ask the question, what are the contents of an XRNAX extract when FA-crosslinking is used?
Third, FA crosslinks everything. That means the connectivity of the network between protein, RNA and probably also DNA is much higher than in the case of UV-crosslinking. This is very consequential for any downstream application. For example, one has to ask the question, if silica enrichment also works on a sponge of protein and RNA?
Our intuition is that very low percentage or short crosslinking could overcome some of those problems by making crosslinks sparse. Anyway, let us know if you found a way to use FA crosslinking for XRNAX. But make sure you have the right controls because it seems tricky to us.