Label IT µArray® Dual Labeling Kit

Figure 1: The Label IT® µArray Dual Labeling Kit. The Label IT biotin and fluorescein reagents are composed of three regions: a biotin or fluorescein label (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 cDNA or rRNA molecules. The covalent attachment of the Label IT reagents to cDNA or cRNA does not alter their structure or downstream hybridization performance, and thus, once labeled, the biotin and fluorescein labeled cDNA or cRNA samples can be pooled and hybridized to gene expression microarrays. Hybridized molecules are then detected using anti-fluorescein antibodies and streptavidin each conjuagated with an appropriate fluorephore.

Figure 2. The Label IT µArray Dual Kit Labels any Nucleic Acid Sample. The Label IT µArray Dual Labeling Kit was used to label mRNA directly for hybridization to duplicate double-stranded cDNA arrays (top), or cDNA and cRNA samples for hybridization to sense-strand oligo arrays (middle, bottom). For each microarray hybridization, the mRNA sample was divided, labeled independently with biotin and fluorescein, pooled, and hybridized to the indicated arrays. All microarray features should be yellow since they were derived from the same sample. Labeled samples were detected using a Streptavdin-Cy™3 and an anti-FL-Cy™5 conjugate.

Figure 3. Direct mRNA Labeling for Gene Expression Analysis. To compare the effect of labeling biases on microarray data, brain and liver mRNA samples were each directly labeled for analysis in each channel and pooled appropriately for a "label-swap" experiment using spotted cDNA microarrays (top). Equivalent gene expression ratios were determined for each gene from both analyses (bottom), indicating the reproducibility of gene expression results and the absence of label incorporation bias. In the label-swap experiment, the average log2 ratio of the Cy™5/Cy™3 signal ratio for each gene is expected to change sign (e.g. from +1 to -1) when samples are labeled for detection in the opposite channel. Each microarray hybridization contained six replicate features per gene and was conducted in duplicate.

Figure 4. Sensitive mRNA Detection: < 10 Copies Per Cell. Sensitivity of detection with the Label IT µArray Dual Labeling Kit was determined from spiking experiments in which a specific quantity of an Arabidopsis mRNA sample was added to HeLa mRNA before labeling. An estimated mRNA copy number of ~10 copies per cell (30 pg of the spiked mRNA in 1 µg HeLa mRNA) can be detected in each channel above non-specific hybridization signal (negative controls). Fluorescent signal detected for the spiked mRNA and negative controls is corrected by subtracting the median local background value; error bars represent one standard deviation.