The many genome sequences currently available hold great promise for biology arid biomedical research. But although DNA sequence information can be used to predict the existence of genes, it does not tell how the gene products function. Evaluating gene expression patterns is a powerful tool in the elucidation of biochemical pathways, in understanding the mechanism of disease, and consequently an important step to drug discovery. Methods used to analyze gene expression include Northern blots, RT-PCR (reverse transcription-PCR) and microarrays. Although a newcomer to the scene, microarrays, such as those offered by Affymetrix, are becoming the preferred method for analysis because they offer the option of analyzing the expression of thousands of genes in parallel. Analysis of gene expression using microarrays involves many steps, some of which introduce biases. We propose to test a microarray-based approach for quantitating mRNA levels which is designed to circumvent several troublesome steps in analyzing gene expression. The proposed method uses a chip array format and an enzymatic on-chip signal amplification. Specifically, the method removes the need (i) to reverse transcribe mRNA; (ii) to amplify signals using PCR or transcription; and (iii) to label target mRNAs or cDNAs.
The specific aims of this Phase I proposal are to fabricate a microarray prototype and identify assay conditions to measure the levels of two specific mRNAs among a pool of human mRNA. Although the proposed method can use both fluorescent and label-free platforms, in Phase I we will focus on label free detection only, using GWC's commercially available SPRimager(r) as the detection platform. The SPRimager(r) uses Surface Plasmon Resonance (SPR) to detect molecular events on the chip surface and therefore does not require any labeling of probe or target molecules. ? ?
