Label IT® µArray Biotin Labeling Kit

Figure 1: The Label IT® µArray Biotin Labeling Kit. The Label IT biotin reagent is composed of three regions: the biotin label (green), the linker (yellow) which facilitates electrostatic interactions with nucleic acids and the reactive alkylating group (blue) that covalently attaches the Label IT biotin 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 cDNA or cRNA samples can be hybridized to gene expression microarrays, detected and analyzed to determine the gene expression profile.

Figure 2. The Label IT µArray Biotin Kit Labels any Nucleic Acid Sample. The Label IT µArray Biotin Reagent 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). Labeled samples were detected using a Streptavidin-Cy™3 conjugate.

Figure 3. Gene Expression Profiles of Labeled mRNA and cDNA Samples. HeLa mRNA and cDNA samples, prepared with the Label IT µArray Biotin Kit generate the same gene expression profiles as cDNA samples labeled with reverse transcriptase incorporation of biotinylated nucleotides. Slightly different quantification of some genes (e.g. A04, C09) with labeled mRNA compared to either method of labeling cDNA may reflect enzymatic replication bias inherent with cDNA sample preparation, and demonstrate the advantage of directly labeling and hybridizing mRNA to microarrays. Relative gene expression values for each labeling method were generated from the average of multiple replicate features from six technical replicate arrays using the same HeLa total RNA sample (data from each microarray hybridization were normalized before averaging).

Figure 4. Sensitive mRNA Detection: < 10 Copies Per Cell. Sensitivity of detection with the Label IT µArray Biotin 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 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.

Figure 5. The Label IT µArray Biotin Kit is Compatible with SuperArray DNA Microarrays. HeLa cRNA (5 µg) was biotin labeled using the Label IT µArray Biotin Labeling Kit, purified, and hybridized to an Oligo GEArray® Human Cancer Microarray (SuperArray Biosciences Corp.) according to the manufacturer’s recommendations. The microarray membrane was developed using chemiluminescent detection. The array shown here resulted from a 5 minutes exposure to X-ray film. The relative expression of the pathway-specific cancer-focused genes is evident.