RNA extraction - nuclear and cytoplasmic fractions - (Jun/09/2006 )
Does anyone know how to succesfully extract nuclear and cytoplasmic RNA without using the columns such as QIAGEN? Something like using Trizol for total RNa extraction. My nuclear fraction is constantly getting contaminated with ribosomal RNA or, rather, I can't get rid of the ribosomal RNA in the nuclear fraction when using columns. I've tried many things like washing the nuclear fraction (pellet) few times in the lysis buffer used to separate the two fractions (cytoplasmic and nuclear), washing in PBS, ect with no luck. Thanks a bunch!
If I want to extract total RNA I use TRIPURE wich combines this agent with chlorophorm, isopropanol and ethanol. I obtain a lot of RNA, much more than with the columns but is total RNA.
Hope I help you
Chomczynski, P., and N. Saachi. 1987. Single-step methof of RNA isolation by acid guanidinium thiocyanate-phenol-chlorofom extraction. Anal. Biochem. 162:156-159
But I just can not find the paper on my messy desk... :-(
since ribosomal RNA is produced in the nucleus, you will always have a certain impurity, maybe you can use this to normalize your experiment somehow. Why living with it when not using it?