Protocol Online logo
Top : New Forum Archives (2009-): : siRNA, microRNA and RNAi

siRNA quantification using UV - (Aug/16/2013 )



I am trying to quantify some freeze-dried siRNA received from a company. I dissolved the siRNA in RNase free TE buffer (pH 7.6) and diluted further in either RNase free PBS or DEPC water to get a UV reading of between 0.2-1.


I find that the readings are very different. The DEPC water diluted siRNA gave a much higher 260 reading (50% more) than the PBS diluted siRNA. Proper baselines were used. The 260/230 and 260/280 ratios did not change, however.


The siRNAs themselves are ~14000 in molecular weight, and there is a modification on the 3' end of the sense with cholesterol linking, and PS modification on the 5' and 3' ends.


I wonder why this is the case and which reading I should use to quantify the siRNA.


Thank you very much!




What reading will you get if you use blank DEPC water and PBS for the measurement?