Tips from the Bench: Transfection Tip

Track Intracellular Nucleic Acid Delivery

Assessing Nucleic Acid Delivery via Direct Nucleic Acid Labeling

A direct method for assessing the success of transfection technique is to label the nucleic acid to track its intracellular delivery. In order to track nucleic acid delivery, fluorophore or affinity tags can be attached to nucleic acid. Ideally, the label should not influence the functionality of the labeled nucleic acid. There are different ways to classify nucleic acid labeling methods:

  1. Chemical labeling technologies are based on reactive groups that bind to nucleic acids. Labeling reagents react with the nucleic acid to chemically attach labels in a non-enzymatic fashion. For example, the Label IT® Labeling Reagents consist of the label, a cationic linker and an alkylating reactive group.
  2. Enzymatic labeling methods are based on the incorporation of label-modified nucleotides during an enzyme-mediated nucleic acid synthesis reaction.

Labeled nucleic acids can be detected in different ways such as:

  1. Direct detection - the labeled nucleic acids contain the detector molecule that will be used for optical, luminescent or fluorescent signal generation, e.g. Cy®3 or rhodamine (Figure 1).
  2. Indirect detection - the labeled nucleic acids contain a label or tag which needs to specifically interact with a reporter-conjugated affinity molecule, e.g. biotin bound by a fluorophore-conjugated streptavidin.
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Measuring Transfection Efficiency

Fluorescence Tracking of Labeled Plasmid DNA Delivery and YFP Reporter Expression. Confocal fluorescence microscopy of COS-7 cells transfected with TransIT® LT-1 and EYFP-Nuc plasmid DNA labeled using Label IT® Nucleic Acid Labeling Kit, CX-Rhodamine. Image panel shows COS-7 cells 1, 4, 8 and 24 hours after transfection with rhodamine-labeled plasmid DNA. Note the changes in labeled plasmid DNA (red) localization from the surface of the cells to a perinuclear location during the time progression.

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