For the Silencer™ siRNA Labeling Kit

Measuring the base:dye ratio

1. Dilute the labeled RNA 5–10 fold in 200 mM MOPS pH 7.5 (adjust the pH with NaOH).
   
   
2. Blank the spectrophotometer with the 200 mM MOPS at 260 nm, and at the maximum absorbance wavelength for the dye (A_dye): 550 nm for Cy3, 649 nm for Cy5, or 492 nm for FAM.
   
   
3. Measure the absorbance of the diluted RNA 260 nm, and at the maximum absorbance wavelength for the dye (A_dye): 550 nm for Cy3, 649 nm for Cy5, or 492 nm for FAM. With these absorbance values in hand, use the convenient base:dye ratio calculator on our web site to do the math explained in Steps 4-6 below.
   
   
4. Since Cy3 and FAM absorb some light at 260 nm (as well as at their absorbance maxima), remove their contribution to the A₂₆₀ reading with the following calculation and the appropriate dye correction factor from Table 1 (below) .
   
   A_base = (A₂₆₀) – (A_dye x dye correction factor)
   
   
5. Calculate the ratio of bases to dye molecules using the following equation:
   
   base:dye = (A_base x extinction coefficient_dye) / A_dye x extinction coefficient_base)
   
   
6. Using values from Table 1 (below) and the corrected absorbance of the labeled RNA from Step 4 above, the concentration of the labeled RNA can be calculated using the following equation:
   
   mg/ml RNA = (A_base x MW_base) / (extinction coefficient_base x pathlength in cm)
   
   Note: Most spectrophotometers have a 1 cm path length; if you don’t know the path length for the spectrophotometer, assume that it is 1 cm.

Table 1. Reference Values for Spectrophotometry Calculations.