cDNA storage stability issues - cDNA storage (Aug/19/2005 )

Hi,
I use RNA extracted from human PBMC and have noticed that my cDNA (stored for over 6 months at -20) has deteriorated. Does anyone know how long cDNA can be stored before it deteriorates? Is there something I can do to prolong the half life?
Thanks,
C

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Hi cDNA is DNA it should be stable enough at -20, RNA on the otherhand is better stored at -80. You could try storing cDNA at -80, can you not carry out your RT-PCR and subsequent protocols quicker, I carry out RT-PCR and then once it is finished i do my RAPD-PCR overnight so i have it the next morning and my RAPD products are then stored long terms, though not as long as six months.

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Hi there
i recently asked a question about RNA from PBMCs on this forum but got no reply so was wondering if you could help.
I am basically isolating PBMCs from whole blood using lymphoprep. Then using a miscroscope i count count my cells and plate them out at various concentrations on a 96 well plate (round bottomed). then after being left overnight i add LPS at 3ug/ml to some of the wells and leave some as controls, then over a period of 4 days i collect the cells in trizol and extract the RNA using the qiagen spin columns., At first my yeilds were poor, and i was only getting about 100ngs of RNA. I was only using 100000 or 300000 cells in eahc of the wells, which i thought would be enough. The second time i ran the experiment i used more cells, from 600000 to 2000000 cells in 24 well flat bottom plates. the yeild i got were much higher - ranging from 400ngs to 1.2ug. This is enough for me to work with as i only have a few assays to run. I just wondered how many cells you used routinely and if you left them to culture overnight or longer before adding the stimulus? some people say they leva eovernight and then tip off the cells that havent adheared and add fresh media - do you do this? do you use special media for PBMCs or just regular RPMI?

sorry for the long winded question -
just loking for a bit of advice
cell culture and RNA work is all a bit new to me

thanks
Jenny

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