Why am I not able to detect my fluorescent labeled siRNA (e.g. FITC) after 24 or 48 hours post-Nucleofection?


Fluorescently labeled siRNA duplexes can be used to analyze transfection efficiency by fluorescence microscopy or flow cytometry.

However, FITC, Rhodamine, or Alexa-488 labeled siRNA oligos should be analyzed 0.5-3 hours post-Nucleofection™. Cy-5 labelled siRNAs can be detected up to 24 hours post Nucleofection™. Likewise, if one is attempting to visualize a label via microscopy, you may have to use a concentration that is much greater (5 µg-10 µg per sample) than that required for functional analysis.

Some labels are also subject to photo bleaching, pH changes, and may exhibit cell type specific effects as well. To minimize FITC-bleaching it may help to wrap the culture plates in aluminum foil immediately after Nucleofection™.

So, it is not possible to both determine efficiency and look for functional knockdown in the same experiment. A better and more cost effective positive control is to use a known sequence to knock down an endogenous housekeeping gene.