这整个坐骑<em>原位</em>杂交协议的讨论,以确保在E8.5 – E11.5日龄小鼠胚胎的基因表达研究的重复性高品质的成果的关键步骤。
整装原位杂交,是一个非常翔实的办法确定胚胎中的基因表达模式。 原位杂交程序冗长,技术上与多个重要步骤,集体作出贡献的最终结果的质量要求,该协议详细描述了优化探针标记和性能的几个关键质量控制步骤。总的来说,我们的协议提供了详细的说明必要的关键步骤,可重复获得高质量的结果。首先,我们描述了一代digoxygenin(DIG)标记的RNA探针通过在体外经PCR产生的DNA模板转录。我们描述了三个关键的质量控制检测,以确定的数量,完整性和辛标记探针的具体活动。这些步骤是重要的生成足够的灵敏度探测器检测内源性的mRNA在整个小鼠胚胎。此外,我们描述的固定和储存E8.5 – E11.5日龄小鼠胚胎原位杂交的方法。然后,我们描述了详细的方法有限蛋白酶K消化杂交条件的细节,杂交后洗涤和核糖核酸酶处理,除去非特异性探针杂交的水化胚胎。 AP -共轭抗体用于可视化探针,并揭示了内源性转录的表达模式。代表实验成功和典型的不理想的实验结果显示。
在本议定书中所描述的方法已经适应从不同来源和整装E8.5 – E11.5日龄小鼠胚胎进行了优化。整个装载在脊椎动物胚胎原位杂交方法最早出现在20世纪90年代初2,5-12。该协议被改编,主要是从非洲爪蟾胚胎 7,8以及鼠标 2,11开发的方法。在我们的协议中,很大的重点放在探头严格的质量评估。仔细注意产生高品质和高比活性的RNA探针在这个协议中最早的步骤确定将…
The authors have nothing to disclose.
这项工作是由国立卫生研究院拨款R21MH082360(BGC)和R01HD056315(NRM)以及佐治亚大学的支持。
Name | Type | Company | Catalogue number | Comments |
---|---|---|---|---|
Digoxigenin-11-uridine-5′-triphosphate | Reagent | Roche | 11209256910 | |
Ribonucleoside Triphosphate Set | Reagent | Roche | 11277057001 | |
Quick Spin Columns for radiolabeled RNA purification | Supply | Roche | 11274015001 | |
T7 RNA polymerase | Reagent | Roche | 10881767001 | |
SP6 RNA polymerase | Reagent | Roche | 10810274001 | |
T3 RNA polymerase | Reagent | Roche | 11031163001 | |
CTP, [α-32P]- 800Ci/mmol 10mCi/ml , 250 μCi | Reagent | Perkin Elmer | BLU008X250UC | |
DNase I recombinant, RNase-free | Reagent | Roche | 4716728001 | |
Urea,Colorless-to-white Crystals or Crystalline Powder | Reagent | Fisher | BP169-500 | |
Gel loading buffer | Reagent | Ambion | AM8547 | |
Hybond-N+, Amersham | Supply | GE Healthcare | RPN82B | |
UV Stratalinker 2400 | Equipment | Stratagene | ||
Diethyl pyrocarbonate | Reagent | Sigma | D5758-100ML | |
Heparin sodium | Reagent | Acros | 41121-0010 | |
hydrogen peroxide 30% in water | Reagent | Fisher scientific | BP2633-500 | |
Gluteraldehyde, 8% EM grade | Reagent | Polysciences | 0/710 | Use with caution according to manufacture’s instructions |
Blocking reagent | Reagent | Roche | 11096176001 | Make into 5% stock and store at -20° C |
Proteinase K | Reagent | Roche | 3115852001 | |
Rnase A | Reagent | Roche | 10109142001 | Make as 10 mg/ml stock and store at -20° C. |
Rnase T1 | Reagent | Roche | 10109193001 | |
Ultrapure Formamide | Reagent | Invitrogen | 15515-026 | |
Levamisole hydrochloride | Reagent | ICN Biomedicals. Inc | 155228 | |
Ribonucleic acid from torula yeast,Type VI | Reagent | Sigma | R6625 | phenol/chloroform extracted several times and precipitated, resuspended in DEPC-dH2O and stored at -20° C |
Anti-Digoxigenin-AP Fab fragments | Reagent | Roche | 11093274910 | |
BM Purple AP Substrate, precipitating. Ready-to-use solution. | Reagent | Roche | 11 442 074 001 | |
4mL, Clear, Closed Top, Storage Vial Convenience Kit | Supply | National scientific | B7800-2 | |
Shake N Bake hybridization oven | Equipment | Boekel Scientific | 136400 | |
Biopsy Sure-Tek | Supply | Fisherbrand | 15-200-402C | |
Paraplast Plus tissue embedding medium | Reagent | Fisherbrand | 23-021-400 | |
Peel-A-Way disposable plastic tissue embedding molds | Supply | Polysciences | 18646A | |
Colorfrost Plus Microscope slides | Supply | Fisherbrand | 12-550-17 | |
Nuclear Fast Red | Reagent | Sigma | N8002 | |
Cytoseal 60 | Reagent | Richard-Allan Scientific | 8310-16 |