Hi,
I am trying to transfect multiple myeloma cell lines with siRNAs using the Amaxa nucleofector. I extract RNA with the TRIzol method 48h after the nucleofection, and I find that while 260/280 ratios are OK, the concentration (50-200 ng/uL) and 260/230 ratios are low (0,6-1) except for the negative control, which looks better (700 ng/ul, 260/280 1.8 and 260/230 1.6). I usually do a ethanol precipitation as a last step and then I let RNAs dry about 30min before I add warm H2O DEPC. I don't know if the low ratio is due to the fact that my cells are dying because of the nucleofection (I find that 50% cells are dead 48h after nucleofection) or because I am not evaporating ethanol correctly.
Should I try to evaporate ethanol better? Or should I try to transfect by other means? Will I see a reduction in mRNA by qPCR extracting RNA after 24h?
Thank you so much,
Ane
Hi Ani.
Wash your RNA pellet with 70% ethanol to remove impurities after ethanol precipitation then proceed for drying step. I hope this will work for you
Gud luck!
Yes, you should evaporate ethanol completely. Any trace amount of ethanol could affect the quality of RNA.
Good luck!
Thank you, I will also try the qiagen kit and see if I impove the ratios...
I have a similar issue. Is there any chance I do something to improve my 260/230 ratio in the same smaples, without doing new ones?
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