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Recovery of Developmentally Defined Gene Sets from High-Density cDNA Macroarrays

Rast, Jonathan P. and Amore, Gabriele and Calestani, Cristina and Livi, Carolina B. and Ransick, Andrew and Davidson, Eric H. (2000) Recovery of Developmentally Defined Gene Sets from High-Density cDNA Macroarrays. Developmental Biology, 228 (2). pp. 270-286. ISSN 0012-1606. http://resolver.caltech.edu/CaltechAUTHORS:20160114-071949663

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Abstract

New technologies for isolating differentially expressed genes from large arrayed cDNA libraries are reported. These methods can be used to identify genes that lie downstream of developmentally important transcription factors and genes that are expressed in specific tissues, processes, or stages of embryonic development. Though developed for the study of gene expression during the early embryogenesis of the sea urchin Strongylocentrotus purpuratus, these technologies can be applied generally. Hybridization parameters were determined for the reaction of complex cDNA probes to cDNA libraries carried on six nylon filters, each containing duplicate spots from 18,432 bacterial clones (macroarrays). These libraries are of sufficient size to include nearly all genes expressed in the embryo. The screening strategy we have devised is designed to overcome inherent sensitivity limitations of macroarray hybridization and thus to isolate differentially expressed genes that are represented only by low-prevalence mRNAs. To this end, we have developed improved methods for the amplification of cDNA from small amounts of tissue (as little as ∼300 sea urchin embryos, or 2 × 10^5 cells, or about 10 ng of mRNA) and for the differential enhancement of probe sequence concentration by subtractive hybridization. Quantitative analysis of macroarray hybridization shows that these probes now suffice for detection of differentially expressed mRNAs down to a level below five molecules per average embryo cell.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1006/dbio.2000.9941DOIArticle
http://www.sciencedirect.com/science/article/pii/S001216060099941XPublisherArticle
Additional Information:© 2000 Academic Press. Received for publication August 24, 2000. Revised September 14, 2000. Accepted September 14, 2000. Published online November 17, 2000. We are grateful to our colleagues Roy J. Britten at Caltech’s Kerckhoff Marine Lab, Professor Ellen Rothenberg, and Dr. Michele Anderson for critical and perspicacious reviews of the manuscript. We thank Rochelle A. Diamond for guidance and help with the purification of transgenic cells by FACS and Xiaohong She and Miki Yun for technical assistance. This research was supported by the National Institute of Child Health and Human Development (HD-37105), the California Sea Grant College Program (NA65RG0477), the Beckman Institute of Caltech, and the Lucille P. Markey Charitable Trust. J.P.R. was supported by an NIH Individual NRSA (GM 18478). C.B.L. was supported by a Walter & Sylvia Treadway Fellowship.
Funders:
Funding AgencyGrant Number
National Institute of Child Health and Human Development (NICHD)HD-37105
California Sea Grant College ProgramNA65RG0477
Caltech Beckman InstituteUNSPECIFIED
Lucille P. Markey Charitable TrustUNSPECIFIED
NIHGM 18478
Walter & Sylvia Treadway FellowshipUNSPECIFIED
Subject Keywords:macroarray; arrayed cDNA library; sea urchin; Strongylocentrotus purpuratus; subtractive hybridization
Record Number:CaltechAUTHORS:20160114-071949663
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20160114-071949663
Official Citation:Jonathan P Rast, Gabriele Amore, Cristina Calestani, Carolina B Livi, Andrew Ransick, Eric H Davidson, Recovery of Developmentally Defined Gene Sets from High-Density cDNA Macroarrays, Developmental Biology, Volume 228, Issue 2, 15 December 2000, Pages 270-286, ISSN 0012-1606, http://dx.doi.org/10.1006/dbio.2000.9941. (http://www.sciencedirect.com/science/article/pii/S001216060099941X)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:63665
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:14 Jan 2016 19:46
Last Modified:14 Jan 2016 19:46

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