Signal Amplification Post Hybridization

Another approach employed to increase sensitivity of microarrays is signal amplification at the post-hybridization stage. One such technique is tyramide signal amplification (TSA) which requires 20-100 times less RNA than direct cDNA labeling (40). This method was originally used in immunohistochemistry and has been an important tool for immunofluorescence microscopy (41,42).

The TSA technique involves the incorporation of a biotin- or fluorescein-labeled nucleotide and its subsequent detection with conjugate reporter molecules after hybridization. The enzyme portion of the conjugate is horseradish peroxidase (HRP), which catalyzes the breakdown of tyramide that results in the deposition of numerous Cy3 labels adjacent to the immobilized HRP. Karsten et al. (40) evaluated the use of the TSA method with archived samples on microarray gene expression analysis. Although they report that the TSA method worked well to amplify the signal from frozen tissue with good-quality RNA, there were still issues of poor reproducibility and reliability especially with formalin-fixed tissue samples. RNA from fixed tissue is generally of poor quality. Partial degradation and modification by proteins or chemicals usually results in disrupted cDNA synthesis with a high rate of misincorporations and short product length. These factors influence the specificity of hybridization, and the TSA method did not reduce their effects (40).

Alternatively, dendrimers (highly branched molecules) can be used to increase the amount of label per nucleotide, and subsequently per labeled cDNA/cRNA molecule (www.genisphere.com). This technology involves dendrimers, each carrying hundreds of fluorescent tags, binding to each hybridized molecule. Yu et al. (43) evaluated both the TSA and Genisphere 3DNA dendrimer labeling systems and reported that the 3DNA system was less time consuming and produced superior and more consistent results. However, hybridizations with less than 2 |xg of RNA produced low and variable spot intensities (43).

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