what happens when a cell gets frozen without cryopreservative - can polysomes and RNA be extracted from such cells? (Aug/15/2006 )
I had earlier freezed some k562 cells in -80 till the conc reached 2X10^8 and tried extracting polysome from them and later proteinase k treated them and phenol chloroform extracted to get RNA, but did no work as the RT- PCR did not show any result
where do you think it went wrong, was it because I used frozen cells?
can frozen cells be used for such purposes?
thanks in advance
you should be able to extract RNA from frozen cells.
In a previous lab, some collegues were collecting supernatants for cytokine ELISA, and were frozing the pelleted cells directly in -80°C to extract RNA later.
If I remember well they were using a kit from qiagen. t was working
well for RNA extraction and protein extraction, i freeze pellets of cells to the -80.
No preservative is needed.
Eventually if cells are stored in liquid, some degradation may occur.
I don't think of a degradation in your case.
err maybe a silly question but what is the pH of your phenol?
you can use pre heated phenol solution that you add directly on pellets. thaw quicker and increases quality.
the pH of the phenol was 4.5
did you run a sample on a gel to check if there is RNA and the pb comes from RTPCR itself?
pH is appropriate. sorry for the question.