The SuperScript® Indirect cDNA Labeling System is an array labeling kit based on proven methods of indirect cDNA labeling, combined with Alexa Fluor® dyes for an all-inclusive, high-efficiency labeling method. The optimized system provides:
- SuperScript® III for high cDNA yields
- Proprietary amino-allyl/aminohexyl nucleotide mixture to increase signal intensity, giving more array positives
- Balanced Alexa Fluor® 555 and 647 succinimidyl esters for improved correlation coefficients
In this indirect labeling method, mRNA or total RNA is reverse transcribed using SuperScript® III RT, incorporating amino-modified dUTP and dATP into the synthesized cDNA. The template RNA is then degraded by base hydrolysis, and the reaction is neutralized with acid. The aminomodified cDNA is then purified to remove unincorporated nucleotides, primers, and buffers. In the second step, the modified cDNA is coupled with the active form of Alexa Fluor® 555 or 647 dye. The labeled cDNA is purified with a low-elution spin column to remove any unreacted dye. The resulting cDNA labeled with Alexa Fluor® dye is ready for hybridization to microarrays (Figure 1).