We are digesting mouse cortices and trying to extract the RNA from microglia for sequencing. We have gotten the isolation working to get good yield of cells (about 200,000 for both cortices) but the RNA after flow sorting is having really low RNA integrity scores. We are thinking that this is because of the sort process and are thinking about using RNALater for preservation of cells prior to sorting (i.e sorting with RNAlater as the buffer!). Has anyone tried this? If so - does the RNALater degrade the fluorophore signals? Any other tips for isolating RNA from sorted primary cells would be appreciated!
How bad is the RIN? This might be one of the experiments where you have to accept RNA degradation and plan your downstream experiments to compensate for it.
The way RNAlater works is that it actually precipitates the RNA inside the cell. It also denatures a lot of proteins. Typically samples get transferred directly from RNAlater to lysis reagent because if you were to (for example) spin the cells out and put them back in an aqueous buffer, the RNA redissolves, the RNases are active again, and nothing was achieved. I don't think it's possible to put RNAlater through a flow cytometer either because of the salt content.
Some people in my lab did have some success a few years ago - I think they got the RIN up to around 6. If I remember correctly they did it by using obscene amounts of recombinant RNase inhibitor. It might be prohibitively expensive to do that using commercially produced inhibitor, but if you cloned an RNase inhibitor into a plasmid and expressed and purified it yourself, it might be doable.
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