We collected urine samples and then keep them in cell frezzing medium at -80. I tried cell sorting by MACS after thawing the cells and it worked (I checked by IF with specific markers and the cell sorting performance is good). But later I tried qPCR for my GOI and housekeepers the Ct values are very bad. I tried the same thing but for fresh urine samples and it works also, the Ct value looks good. I think there must be something wrong for the samples storage.
May I ask can anyone give any advice? Thanks!
Hi Hang Yan,
Somehow the integrity of the nucleic acid is affected during the freeze and thaw cycles. Make sure that the cells are being frozen properly (follow all the steps on how to freeze cells) and also follow steps for thawing.
If you want to use the cells immediately for MACS, then try following protocol.
This is thawing medium and acclimatizing protocol that was posted by Sevil KÖSE
Thawing medium: Media (e.g. DMEM-LG), 1%PEN/STREPT, 0,1%DNase (10mg in 10mL DMEM-LG for DN25 sigma product)--> Use media that is appropriate for your cells of interest
Protocol:
Good luck!
Best regards
Savita
I suddently have an idea that after I get cell pellet of uirne then directly add RNA Later and mix well. I put them in 4 degree since I read that the RNA later can make the RNA to be stable at 4 degree for 1 month. And in 1 month I definitly will have finished cell sorting. But the problem is I still need to sort the cell by MACS and later I need both DNA/RNA. So I am not sure how RNA later will influence the cell sorting and DNA quality? I can also just have a try but just curious. Do anyone has this experimence?
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