Abstract
Gene indices with many thousands of entries have been constructed by tag sequencing of randomly selected cDNA clones (1–7) and are widely available in repositories, such as the dbEST database (16). As more and more genes are identified, efforts are redirected towards understanding the control of gene expression that occurs in a strictly ordered time and cell-dependent fashion. Expression measurements often constitute a first step in this direction, and can be performed on a reasonably large scale using highly parallel hybridization methods. Hybridization methods, using complex probes and large arrays of targets, derive their power from the fact that each individual experiment provides a very large amount of information. Unrivaled for large-scale measurement of gene expression, these methods are based on the hybridization of complex probes with high-density colony cDNA (or PCR product) filters followed by quantitative measurement of the amount of hybridized probe on each colony. There are three key elements indispensable for accomplishing this task: the robot (for spotting colonies), the phosphoimager (for analyzing exposure), and the image analysis software (Fig. 1).
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Notes
- 1.
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Unfortunately this very useful resource is not completely error-free: Discrepancies in the correspondence between physical clones and tag sequences range from 10 to 20%, imposing expensive and time-consuming verification when these entities are used as reagents
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Bernard, K., Granjeaud, S., Victoréro, G., Nguyen, C. (2000). Transcriptional Expression Analysis of T-Cell Activation by Multiplex Messenger Assay (MMA). In: Kearse, K.P. (eds) T Cell Protocols. Methods in Molecular Biology™, vol 134. Humana Press. https://doi.org/10.1385/1-59259-682-7:337
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DOI: https://doi.org/10.1385/1-59259-682-7:337
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