title: A non-enzymatic microsurgical dissection technique of mouse embryonic tissues for gene expression profiling applications.
abstract: With the increased use of gene expression profiling to identify molecular regulators of cellular and developmental mechanisms, developmental biologists face a new challenge in dissecting tissues without cross-contamination or change in RNA profile, and with intact RNA integrity. We have developed a technique that overcomes these problems. We took the dissection of rudimentary mouse embryonic mammary glands as an example, as these structures are particularly difficult to separate from their contiguous ectoderm and strongly adhering mesenchyme. Contrary to conventional enzymatic tissue-separation methods, we blocked transcriptional activity prior to dissection and protected RNA from degradation during dissection, by the use of RNAlater. While RNAlater dehydrates specimens so severely that it interferes with visibility and clean dissection of organs or tissues, we established rehydration conditions that in fact facilitated tissue separation and shortened dissection time to about 10 minutes. The extracted RNA had an excellent quality, rendering it perfectly suitable for transcriptional profiling. Visual inspection of separated tissues and tissue specific gene expression analysis by microarray and RT-PCR confirmed that the tissues were separated with minimal or no cross-contamination. We show that this dissection method can be applied to a broad variety of organs, and that the tissue is still amenable to protein detection. In conclusion, this is a rapid, cheap and effective non-enzymatic tissue separation method which greatly facilitates the exploration of molecular mechanisms in organ formation.
authors.affiliation: Institute of Molecular and Cell Biology, A STAR (Agency for Science, Technology and Research), Singapore.
authors.forename: Li
authors.initials: L
authors.lastname: Sun
authors.forename: May-Yin
authors.initials: MY
authors.lastname: Lee
authors.forename: Jacqueline M
authors.initials: JM
authors.lastname: Veltmaat
mesh_terms: D000818:Animals; D020405:Dermis / Q000196:embryology; D005260:Female; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D008321:Mammary Glands, Animal / Q000196:embryology; D051379:Mice; D008810:Mice, Inbred C57BL; D008866:Microsurgery / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D012313:RNA / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution
chemical_list: D012313:RNA; D012260:Ribonucleases