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.

Sox9 regulates cell state and activity of embryonic mouse mammary progenitor cells

“…Embryonic mammary tissues (embryonic mammary primordial epithelium (MPE), embryonic mammary stroma (includes MM and FPP), and surface epithelium (Epi) were microdissected from E12.5 B6 mice following the protocol described in ref. 46 . Total RNA from cells and mammary tissues were extracted using RNeasy Plus Micro Kit and RNeasy Mini Kit (74034 and 74104, respectively, Qiagen, Manchester, UK), RNA concentration and genomic DNA (gDNA) contamination were determined using Qubit 2.0 Fluorometer and accompanying kits (Thermo Fisher Scientific, Waltham, MA, USA).…”

Investigating Molecular Mechanisms of Embryonic Mammary Gland Development by Bead-Implantation in Embryonic Flank Explant Cultures – A Protocol

“…4A movie showing embryo dissection (step 2) is available online at http://www.ijdb.ehu.es/data/11/113424ls/IntJDevBiol-113424ls-SuppMovie1.mp4 [13], and needs to be downloaded (by opting “save link as…”) for viewing. Note that for this protocol, cleaning of surfaces with RNase-away is not necessary, and after dissection, embryos should NOT be collected in RNA-later, but in sterile DPBS.…”

“…5A movie showing flank dissection (step 4) is available online at http://www.ijdb.ehu.es/data/11/113424ls/IntJDevBiol-113424ls-SuppMovie2.mp4 [13], and needs to be downloaded (by opting “save link as …”) for viewing. Note that embryos in the movie were stored in RNA-later and thus look wrinkled/shriveled compared to the fresh embryos one uses for culture experiments.…”