I have been trying to isolate good quality from mouse spleen for a while, but my RIN scores are always around 7 or below. My workflow typically includes harvesting the spleen in sterile pbs, sectioning it into 500um slices, staining overnight (rpmi and 1% bsa + antibody), imaging, dissociating in pbs, performing FACS (sterile method), collecting the pellet (centrifuging sorted sample), then lysing in trizol for rna extraction. I know that there are many steps that could be affecting the quality, but I always make sure to include an unprocessed control that is dissociated in trizol immediately after sectioning, and still the RIN is not satisfactory. There was one time I was able to obtain a RIN of 8.3, but I am wondering if there is anything specific I need to do to get reproducibly high RIN scores. I think my processing steps are not affecting the quality that much since the RIN scores of my unprocessed sample and processed samples are usually in the same range. Any advice would be valuable!
An option to improve RNA quality might be to add an RNase inhibitor to the PBS for spleen harvesting and sectioning.
Sabine Strehl thanks for always helping me out! Ive been adding an rnase inhibitor to my staining solution but perhaps i should increase the amount i use and start adding it to the pbs too (though usually when im using pbs with cells i get rin scores as high as 9+). I think I also might be using too much trizol to lyse my cells so next time im going to try adjusting the volume according to the cell count.. though i doubt thats the case either! im getting pretty decent 28s/18s ratios even after processing so i think there might be protein contamination from somewhere. im going to try adjusting all these next time but otherwise think it might be worth it to try using another kit.
Thanks again for the help i really appreciate it!
It might be that during sectioning RNases are released from the cut cells. In our hands RNA isolation with Trizol yields RNA with high RIN values, I would not necessarily use another RNA isolation kit.
Sabine Strehl hmm you think so? do you know of any way to reduce this?
Just a thought...
That's why I suggested to add RNAase inhibitor to the PBS for spleen harvesting and sectioning.
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