Importance

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Abstract

Quantification in a straightforward manner in an mRNA expression analysis is accomplished by using biotinylated oligo d(T) and coupling to a solid support (e.g., streptavidin-derivatized magnetic beads). Labeled targets specific to certain mRNAs of interest that are to be identified and quantified are added to a biological sample containing the mRNAs and biotinylated oligo d(T). The beads are preferably added after hybridization, but may be earlier added. Following attachment to the beads, unbound and non-specifically bound targets and non-mRNA material are removed by successive stringent washings. All the mRNA material is then eluted from the beads by subjection to conditions that separate the poly A and the oligo d(T), and the original mRNAs are then degraded by basic hydrolysis to leave single-strand synthetic targets, which are then hybridized to their specific probes suitably carried on a microarray. The probes and the targets are of synthetic designs, and because all affinity reactions, including hybridization reactions between the targets and the mRNAs, are finalized when the mRNA is bound to the solid substrate, purification of adducts can be performed by simple washings in combination with magnetic separation, avoiding any potentially laborious and time-consuming column separation, filter preparation, centrifugation or preciptitation.

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First Claim