RNA Isolation with RNeasy - (Dec/05/2005 )
We isolate total RNA from Lymphocytes using the RNeasy Kit from Qiagen. Prelimiary tests worked well (amount: about 750 ng RNA per 2 x 106 cells). Cells are treated as follows: isolation in EDTA, buffy coat, stain and FACS sort, before we freeze the cells we dissolve cells in 350 ul RLT buffer & beta mercaptoethanol and homogenize them using QIAshredder. The cells are then stored at -80°C. Before isolation the samples are equilibrated to room temperature for at least 15 minutes. From these frozen cells I don't get more than 150 ng RNA whereas if I isolate RNA from samples that are not frozen I still get sufficient RNA. I really can't see why it's not working with frozen samples anymore.... I'm happy to take your suggestions, thanks
one thing which i would like to ask from you how are you equlibrating your stored RNA samples to room temperature. It must be done throughout in ice....... In your case you are promoting RNase activity.
yep. go straight into the frozen pellet with the RLT
The homogenized cells are stored at -80°C in RLT, which is recommended by QIAGEN. Before isolation you have to equilibrate the sample to room temperature since RLT forms salts at temperature below 18°C.
In that case I would try adding the ethanol directly to the frozen lysate then letting it equilibrate
i don't use the qiagen kit but tri family. It's based on phenol and i noticed i got far better results if i preheat at 50° tri reagent or trizol and add it directly on the frozen pellet. The thaw is quicker and RNAses can not act long time, which is probably what occurs when slow defrosting before lysis.
Try snap freezing the pellet. RNAses are activated as the cell realizes its hitting unfavorable conditions- as it gets cold. Snap freezing doesn't give the RNAses a chance to do any degredation.