急求帮忙怎么分离外胚层，难道直接将5天的胚胎直接消化提取RNA

chenchunlei

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老板想让我把外胚层的marker做下定量，我想知道怎么分离外胚层，难道直接将5天的胚胎直接消化提取RNA，不知道可不可以

zzzhouzhong

偶然看到一篇paper，07年的nature，Derivation of pluripotent epiblast stem cells from mammalian embryos- `2 a* P3 s/ ?
http://www.nature.com/nature/jou ... ll/nature05950.html
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For derivation of EpiSCs, the late epiblast layer was dissected intact from pre-gastrula stage mouse [E5.75, (B6XCBA)F1 mated inter se; E6.5, NOD mated inter se] or rat [ E7.5-7.75 Wistar or Sprague Dawley] embryos after 15-20 min treatment at 4°C in Cell Dissociation Buffer (Gibco). Dissection was accomplished in Calcium and Magnesium free Flushing and Handling Medium using glass needles to completely separate the epiblast layer from the extraembryonic tissues. Epiblast layers were then placed in chemically defined medium (CDM)5, supplemented with Activin A (20ng/ml, RandD systems) and FGF2 (12 ng/ml, RandD systems). Every 4-5 days, cells were harvested using 5 mg/ml collagenase IV (Invitrogen) then plated into plates (Costar) pre-coated either with fetal bovine serum (FBS) or with 15 μg/ml of human Fibronectin (Chemicon) for 20 min at 37C and then washed twice in PBS.