EtBr vs. spectrophotometric RNA quantitation

[Rafael Perez Vicente]

Dear Netters,


We always found lanes with significant differences in the EtBr fluorescence 
intensities after loading apparently the same amount of RNA per lane, from 
different plant RNA samples. RNA concentration of each sample was accurately 
determined by measuring the absorbance at 260 nm. 

A certain amount of carbohydrates, that vary from sample to sample, is the 
only relevant contaminant observed in our RNA. 

My question is: Are the carbohydrates expected to interfere the measurement of
the absorbance at 260 nm? If the answer is not, are then the carbohydrates 
interfering the binding of EtBr to RNA and thus modifying the fluorescence?

In other words, what should I consider to be the right measurement of the RNA 
in this conditions, the spectrophotometric one, the EtBr one, none of them.

Is there another method for RNA quantitation not influenced by 
carbohydrates ?



I imagine these are old questions for many of you but I have not got the 
answers yet

Thanks a lot for your time and advice

		  


Rafael Perez Vicente
Div. Fisiologia Vegetal
Fac. Ciencias. Univ. Cordoba
14071-Cordoba
SPAIN

Phone: (57) 218610
            218611

Fax: (57) 218606