I am using the hanging drop method to develop individual differentiated adipocyte spheroids from the stromal vascular fraction of adipose tissue. With our current timeline they end up 100-300um. My problem is, when I remove the spheroids from the matrigel droplet with ice cold 1x PBS, I pool multiple spheroids from the same treatment into one tube for RNA extraction, and the spheroids float and will not pellet when I centrifuge. They float, and I cannot remove the excess PBS without aspirating a spheroid. I’ve tried 300x g, 500x g, even 1000x g for 15 min RT. I don’t want the spheroids to lyse in the PBS, I want to remove the PBS so I can add lysis buffer. Too much residual PBS is what I think is contributing to my low RNA concentrations (1.1 - 6ng/ml for 20 pooled spheroids). I am using the micro RNeasy kit from qiagen, including the carrier RNA they provide for low RNA samples. This picture is after 1000x g centrifugation for 15 min. Any help or advice would be much appreciated!
I would guess that the adipocyte spheroids are the a similar density as the PBS and that is why they will not pellet. I would try to collect them on a filter either by vacuum or by centrifugation on one of the protein concentrating filters with a high molec wt cutoff and then you can extract the RNA from the filter. You just need to make sure the filter is compatible with your RNA extraction protocol
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