Hello everyone!!!!
I have been carrying out co-cultures with A. fumigatus and macrophages. The problem arrives when I try to extract fungal RNA, because I also extract cell RNA and sometimes I get degraded RNA samples. Before RNA extraction I treat cells with DEPC H2O to lyse them.
Have any of you experienced this problem?
Thank you in advance,
Best regards
Macrophages have a high amount of RNases... the DEPC H2O treatment (if you mean DEPC treated water and not actual DEPC in water) will not inactivate the RNases in your samples (DEPC-treated water is thus treated to inactivate any RNases in the water itself only). DEPC itself, if not autoclaved thoroughly will adduct nucleic acids as well; very undesirable; try to use other forms of nuclease-free water. You should instead, lyse the cells with TRizol and extract using that method. Place 2 to 10 million cells into 1 mL of Trizol, homogenize immediately and proceed from there. The Trizol reagent will inactivate any RNases present. Lysing cells in 'water' will allow RNases to degrade your RNA; which is to be avoided.
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