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 (5µg-10µg 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.