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?