Genes that are differentially expressed both in time and space are the basis for how single cells, through the process of embryonic development, give rise to animals with an extraordinary diversity of cell types. As a first step in understanding differential gene expression, many researchers seek to identify those genes whose transcripts are temporally or spatially restricted to particular cells, tissues, or embryonic stages. Although there are a variety of methods suitable for identifying moderately to highly expressed genes, the isolation of the most interesting class of mRNAs, those that are not abundant, but that may be cell-or tissue-specific, remains the most difficult task.
Several basic types of methods have been employed to identify low-abundance, tissue-specific transcripts. The more classical differential hybridization techniques (e.g., 1) are mostly limited to the detection of moderately abundant transcripts representing >0.05% of the mRNA population (2). Subtractive hybridization techniques can increase the detection sensitivity by 10- to 100fold and make the identification of quite rare genes possible (2). A specialized form of subtractive hybridization, the "Gene Expression Screen" (3), can detect both upregulated and downregulated transcripts. A number of protocols have been devised in recent years to simplify and expedite the process of transcript identification by subtractive hybridization (4-8). Here we present a comprehensive set of methods that have proven quite successful in our laboratories and that may serve as an entry point for future refinement.
In the following protocol, we utilize the commonly available and widely used phage vector ^ZAPII, since one can produce oriented libraries in phage or
From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ
phagemids, and subsequently utilize the libraries to produce essentially unlimited quantities of sense or antisense RNAs for subtraction and screening. Furthermore, Stratagene (San Diego, CA) and other suppliers provide a number of high-quality, premade cDNA libraries that can save considerable time if one happens to exist for the tissue of interest. Recent advances in vector technology and hybridization allow one to produce subtracted probes and libraries even when starting material is quite limited.
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