Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research.
Here we report a novel design of linker primer that allows one to differentially amplify long tracts (average 3.0 kb with size ranges of 1-7 kb) or short DNAs (average 1.5 kb with size ranges of 0.5-3 kb) from a complex mixture. The method allows one to generate cDNA libraries enriched for long transcripts without size selection of insert DNAs. One representative library from newborn kidney includes 70% of clones bearing ATG start codons. A comparable library has been generated from 20 mouse blastocysts, containing only ∼40 ng of total RNA. This universal PCR amplification scheme can provide a route to isolate very large cDNAs, even if they are expressed at very low levels.[The sequence data described in this paper have been submitted to the GenBank data library under accession numbers BG060207-BG062928.]A catalog of genes in the form of cDNA clones is the complement to the sequence of genomes, providing not only the confirmation of predicted gene structures, but also the materials for cDNA microarrays and for functional analyses or proteomics (Takahashi and Ko 1994;Lennon et al. 1996;Marra et al. 1999;Strausberg et al. 1999;Tanaka et al. 2000). Because many genes apparently are expressed only at limited times and places during embryogenesis (Ko et al. 2000), a complete cohort requires cDNA libraries from microdissected tissues and small numbers of cells from developing embryos. Because they require large amounts of RNA, conventional cDNA construction methods are precluded for such purposes. The problem is especially acute for full-length cDNA libraries, which require 10-50 µg of polyA+ RNA, equivalent to milligram amounts of total RNA (Maruyama and Sugano 1994;Edery et al. 1995;Carninci et al. 1996Carninci et al. , 1997Carninci et al. , 2000Suzuki et al. 1997Suzuki et al. , 2000.PCR amplification can permit the use of smaller amounts of RNA (Belyavsky et al. 1989), but presents the technical dilemma that it customarily yields a shorter average insert size (e.g., ∼0.5 kb [Peterson et al. 1998] and ∼1.5 kb [Ko et al. 2000]), too short to recover many full-length cDNAs. While working on various linker designs and PCR conditions, we serendipitously found a condition that overcomes size limitations. Here, we report a novel design of linker primer that allows one to differentially amplify long cDNAs or short cDNAs from a complex mixture. RESULTS AND DISCUSSIONTo recover novel genes from mouse embryos, we have been using a PCR-amplification method to construct cDNA libraries. In the original protocol (Takahashi and Ko 1994), doublestranded cDNA mixtures were ligated with a lone linker (LLSal3 [Ko et al. 1990]; Fig. 1A) and amplified by Taq polymerase (Fig. 1B). The resultant cDNA libraries have relatively short inserts (average 0.8 kb, range 0.5-2.5 kb). Subsequent introduction of new enzymes and long PCR methods (Barnes 1994;Cheng et al. 1994) dramatically increased the size of recovered DNA inserts (average 1.5 kb, range 0.5-3.0 kb), and such libraries have been used for a large-scale expressed sequence tag (EST) pr...
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