First , sticky cells(e g. AGS cell line) were isolated using trypsin-EDTA(5mM) then cellular RNAs were extracted using RNAXplus, However, no band was observed on agarose gel! Thank you in advance for any help.
adding 0.1% Tween 20 to all buffers markedly increased the cell number after
each washing step
Hi Mirzababaei
thanks, but first I need to properly isolate the cells then to increase their number.
I wonder whether I clearly understand the expression "sticky cell line". if you mean that these cells are tightly attached to the culture surface, then it is probably the reason why you end up without much RNA in your preps. I say this because you might be using a longer incubation time with trypsin, which can easily shear proteins on cell surface and even damage some cells, making that you loose them during the wash steps.
My question is to know whether you really need to isolate the cells. If not, just wash them in situ with PBS and directly lyse in the culture well/dish with the lysis buffer of your kit. I use this procedure in several cell types and it works pretty well, although with the GenElute total RNA extraction kit from Sigma.
Wishing you success,
Regards
Hi Josué Mfopou K.
Thank you very much, and I mean adherent cells. Yes, these cells are tightly attached to the culture surface.
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