Label IT^® Nucleic Acid Labeling Kit

The Label IT^® Nucleic Acid Labeling Chemistry Enables a Simple Protocol. The Label IT^® chemical labeling reagents are composed of three regions: the label (fluorophore or hapten) (green), the linker (yellow) which facilitates electrostatic interactions with nucleic acids and the reactive alkylating group (blue) that covalently attaches the Label IT^® reagent to any reactive heteroatom within the nucleic acids. Attachment of the Label IT^® Reagents to nucleic acids does not alter the structure of the nucleic acid or affect downstream hybridization performance, and as such, nucleic acids labeled using the Label IT^® Reagents can be employed in multiple applications as defined by the researcher.

Fig. 1 | Non-destructive Direct Labeling of Nucleic Acid. Mouse total RNA was labeled with Label IT^® Cy^®3 (B) electrophoresed and analyzed alongside unlabeled total RNA (A) on an Agilent RNA 6000 Nano chip. RNA Integrity Number (RIN) is approximately 9.0 for both labeled and unlabeled samples.

Fig. 2 | Labeling Density can be Easily Controlled with the Label IT^® Reagents. Plasmid DNA was labeled with increasing ratios (w:w) of Label IT^® CX-Rhodamine to DNA. The extent of labeling was estimated, and normalized to the amount of recovered DNA (# labels/100 base pairs), using two different criteria: fluorescence intensity of the attached rhodamine (excitation at 585 nm, emission at 610 nm), and visible absorbance at 585 nm. The significance of quenching, at the higher labeling ratios, becomes apparent in the fluorimetric estimations of labeling density.

Fig. 3 | Label IT^® MFP488 Labeled Plasmid DNA is Less Sensitive to Photobleaching as Compared to Label IT^® Fluorescein Labeled Plasmid DNA. (A) Plasmid DNA was labeled with Label IT^® MFP488 or Label IT^® Fluorescein. Purified DNA was transfected into HeLa cells using the TransIT^®-LT1 Transfection Reagent at a 3:1 reagent-to-DNA ratio (vol:wt). Two hours post transfection labeled DNA was visualized using a 63X oil immersion objective on a Zeiss Axiovert Inverted Fluorescence microscope. Images were captured before and after a 2 minute photobleaching period from a FITC light source. (B) The ratio (X-axis) of the brightness of individual puncta was calculated by dividing the photobleached value for fluorescence brightness by the value obtained from non-photobleached values for four separate images using Image J software (NIH), the average and standard deviation are shown. Photobleaching was also measured after 5 minutes for Label IT^® MFP488 labeled plasmid DNA and the signal was still 4-fold higher than Label IT^® Fluorescein labeled plasmid DNA at 2 minutes (data not shown).

Fig. 4 | Label IT^® MFP488 Labeled Plasmid DNA is More Resistant to the Effects of Low pH as Compared to Label IT^® Fluorescein Labeled Plasmid DNA. Plasmid DNA was labeled at a low density, 1 label per 65 bp, using Label IT^® MFP488 or Label IT^® Fluorescein Nucleic Acid Labeling Kits. Purified DNA was subsequently diluted in various pH buffers (50 mM HEPES, pH 8, 100 mM MES, pH 5.4, and 100 mM MES, pH 3). Fluorescence was measured using a Tecan Genios Fluorometer and normalized to the respective control measured at 50 mM HEPES, pH 8.