Name: mouse adipose tissue collection and processing for rna analysis
Uploaded: 2018-01-31T14:00:00.000+00:00
Duration: 11 min 13 s
Description: Watch this Scientific Journal Video about Mouse Adipose Tissue Collection and Processing for RNA Analysis at JoVE.com

这项工作得到了加拿大糖尿病患者的支持。

在该程序中使用的小鼠的护理符合加拿大动物保护委员会规定的实验动物的护理和使用标准。所有的程序都由大学动物保护和使用委员会在密友研究中心批准。
1. 雄性小鼠尸检和脂肪组织的收集
<ol><li>根据学习目标制作实验组。本研究从两组雄性小鼠的 C57Bl/6 背景 (n=12-14/group) 中采集样本。确保在这里使用的小鼠有12-15 周的年龄, 并且在12小时的光照/暗循环中保持正常 (N) 或高脂肪 (HF) 饮食和水的ad 随意, 为期10周,11。</li>
	<li>通过将鼠标放置在一个密封的腔中, 在10分钟内接触到 6% CO2 , 安乐鼠标。</li>
	<li>执行心脏穿刺, 慢慢插入一个1毫升注射器安装与 26G 1/2 宝针刚刚在动物的胸骨左侧, 并慢慢拉动活塞, 以消除大部分动物的血液。确保注射器与动物的身体平行。如果血液不进入注射器, 在拉动活塞的同时慢慢旋转注射器。 注意:成功的心脏穿刺 (0.8 和1毫升血容量) 将减少脂肪组织的血液污染。</li>
	<li>将鼠标放在背上, 用22G 针将每个爪子固定在垫子上, 如图 1所示。</li>
	<li>用纱布, 用酒精摩擦老鼠的皮肤, 从颈部一直到生殖器官, 通过单向运动, 这样可以确保头发保持扁平, 减少脂肪组织样本的毛发污染。没有脱毛。</li>
	<li>使用干净但不消毒的镊子和外科剪刀, 提高生殖器官附近的中央皮肤, 并做一个小0.3 毫米切口。</li>
	<li>将剪刀水平插入到打开的孔中, 并将腹部的皮肤从腹壁中分离出来。 注意:这是通过在皮肤和腹壁之间的空间的钝解剖, 而不是通过切割在这个空间发现的膜。</li>
	<li>使用镊子, 提高中央皮肤附近的打开孔, 并削减皮肤的式阿尔巴, 直到你靠近肋骨笼 (〜4厘米取决于鼠标大小)。 注意:避免切割腹部肌肉, 因为这可能暴露内脏脂肪组织的空气和导致干燥的脂肪垫, 这可能会改变完整性的组织及其内容。</li>
	<li>从肋骨中间, 将皮肤朝右手臂的2厘米方向切开。从生殖器官附近, 将右后肢上的皮肤切成2厘米。</li>
	<li>伸展开的皮肤的一个角落, 用22G 针把它钉在垫子上。与另一个角落重复。 注意:暴露在皮肤上的脂肪组织是腹部皮下脂肪 (图 1A)。</li>
	<li>通过钝的解剖和切割它从皮肤上使用的外科剪刀的位置尽可能平坦的皮肤。一旦收集, 将在预先确定的铝箔的流体流量。 注意:收集或污染脂肪组织与皮肤或头发将改变样品质量和 RNA 产量由于 RNases。如果花太多时间收集脂肪组织, 样品也可以干燥, 也可以改变样品的质量。</li>
	<li>在动物的左侧重复步骤1.9 到1.11。将左、右脂肪垫放入铝箔中, 并将样品冷冻在液氮中。</li>
	<li>要进入内脏脂肪组织, 用干净但不消毒的手术剪刀和钳沿式阿尔巴切割腹部肌肉, 在〜4厘米, 根据鼠标大小, 从生殖器到肋骨笼。然后做一个切口在腹部肌肉从生殖器往后面的老鼠在 ~ 2.5 cm 有通入腹部器官的边。</li>
	<li>生殖器官周围的脂肪是围性腺脂肪 (PGF) (图 1B)。当 PGF 附着在附睾、睾丸和输精管上时, 小心地将左脂肪垫从这些结构上分离出来, 并放在预先识别的铝箔上。对右手边重复。一旦两个 PGFs 收集, 冷冻样品在液氮。</li>
	<li>从左手边开始, 通过将肠道从腹腔移到右手边来暴露肾脏。围绕肾脏的脂肪组织是围肾脂肪 (重复脉冲) (图 1C)。重复脉冲也围绕肾上腺 (图 1D)。</li>
	<li>在收集脉冲前分离和切除肾上腺。这可能是单调乏味的, 因为它们只是比脂肪组织的一点点的粉色。 注意:在这一步中, 肾脏不应被移除, 因为血液可能会污染脂肪组织, 使它更难以识别这个脂肪垫内的肾上腺。</li>
	<li>从背部的肌肉壁中分离出脉冲, 以切断输尿管、肾动脉和静脉, 从而将重脉冲和肾脏与身体其他部分分开。通过切除肾囊和切除肾脏来分离肾脉冲。重复频率脉冲保持附在胶囊。</li>
	<li>对右手边重复步骤1.15 和1.17。在铝箔中放入两个重复脉冲, 并将样品冷冻在液氮中。</li>
	<li>重复步骤1.2 至1.18 的所有小鼠要收集的过程。</li>
	<li>在这一点上, 进行脂肪组织研磨或储存样品在一个-80 ° c 冰箱后提取。样品可以稳定地被存放在-80 ° c 几年4。</li>
</ol>2. 用于 RNA 分离的地面白脂肪组织的制备
注意:每一个步骤都要戴上手套, 因为皮肤含有 RNases, 可以促进 rna 的降解, 因此, 分离后的样品质量和 rna 的产量都会改变。
<ol><li>准备和识别三无核糖核酸1.5 毫升锥形螺旋帽管每只老鼠, 一个为每种类型的白色脂肪组织, 并把它们放在一个架子上。 注意:肥胖动物可能需要额外的管, 因为它们增加了脂肪的质量。这是特别正确的, 为7管为一只老鼠获得了。</li>
	<li>用70% 乙醇清洁工作台, 并在表面上放一张干净的新的台式纸。</li>
	<li>脂肪组织是否新鲜地从小鼠收集或预先储存在一个-80 ° c 冷冻库, 收集所有样品在一个液态氮填充容器, 直到程序开始。</li>
	<li>将砂浆、杵和刮刀放在液氮容器中, 将其冷冻。当液氮停止 "沸腾" 时, 可以启动脂肪组织的研磨。 注意:从这一步, 砂浆, 刮刀, 锥形管和脂肪组织需要保持冷藏在整个过程中。任何热量都会融化脂肪组织, 使其难以提取 RNA。此外, 如果使用陶瓷砂浆和杵, 你可以使用干冰和/或液氮来保持它的冷。</li>
	<li>将所有核糖核酸的1.5 毫升锥形螺旋帽管准备在2.2 步, 在干冰冷却下来。</li>
	<li>为了避免手上的将, 用几层棕色的纸把砂浆从液氮中抬起, 放在工作台上。</li>
	<li>使用镊子收集从液氮的脂肪组织样本, 并把它放在砂浆。迅速打开铝箔, 并把样品放在砂浆的中心。 注意:如果脂肪组织样本太大, 如肥胖小鼠的情况下, 用拇指在铝箔的小零件中分解样品, 然后分别粉碎每个部分。</li>
	<li>用几层棕色的纸把杵从液氮中抬起, 然后放入砂浆中。用棕色的纸拿杵, 用锤子击打杵的顶端一两次。用旋转的动作将杵压在迫击炮上, 研磨样品, 直至获得细粉。 注意:此步骤对于获得足够的 RNA 隔离至关重要。事实上, 存在较大的碎片会阻碍 RNA 的分离和降低产量。</li>
	<li>一旦获得了细粉, 取出杵和拾起从液氮的刮刀, 并使用它转移到相应的预冷1.5 毫升锥管样品。 注意:不时地将刮刀浸回液态氮, 以防样品熔化。此外, 保持锥形管在干冰总是防止样品解冻。在足够的 RNA 产量和数量上, 需要一个充满地面样品的锥形管, 大约2/3 容量 (约1毫升)。</li>
	<li>一旦填充到约1毫升, 关闭管, 并把它放到其他液体氮填充容器。 注意:多余的样品可以放在单独的预冷核糖核酸1.5 毫升锥形管, 并储存在一个-80 ° c 冷冻后使用。</li>
	<li>用褐色的纸擦拭砂浆、杵和刮刀, 以避免携带到下一个脂肪组织样品中。把杵和刮刀放回液氮中冷却。即使棕色纸不是核糖核酸的, 我们还是用一包新开的牛皮纸为每一天的 RNA 隔离。</li>
	<li>在下一个示例中重复步骤2.4 到2.11。</li>
	<li>一旦所有样品被处理, 开始 RNA 隔离或存放样品在一个-80 ° c 冰箱为以后使用。 注意:为了防止管子爆裂, 通过将样品箱完全浸入液氮容器来冷冻。当冷却足够的时候, 从盒子中除去多余的氮, 让它漂浮在液态氮的顶端。将样品放入盒子中, 放入-80 ° c 的冷冻柜中。</li>
</ol>3. RNA 与地面脂肪组织的分离
警告:苯酚溶液对皮肤有害。穿上实验室大衣、手套、安全眼镜, 并在化学罩下执行程序。分步流程图如图 2所示。
<ol><li>准备和识别两套无核糖核酸1.5 毫升锥形管, 并把它们放在一个架子上。</li>
	<li>无论脂肪组织是新的地面或预储存在一个-80 ° c 冰箱, 收集所有样品在一个液态氮填充容器, 直到开始的程序, 这是主要在室温下完成。</li>
	<li>用镊子从液氮中选择一个地面脂肪组织样品, 并将其放入管架中。该管应含有〜1毫升的粉末, 这相当于约100毫克的组织粉。</li>
	<li>慢慢打开管子 注意:由于氮气的压力会从试管中逸出, 所以在室温下关闭封闭管或太快打开管盖可能会导致样品丢失或受伤。</li>
	<li>在样品中加入500µL 苯酚溶液 (1:2 比苯酚溶液: 组织粉)。</li>
	<li>关闭管的盖子和漩涡的最大速度约5秒. 慢慢地, 小心地打开管子, 释放来自氮气的压力 (从管子里出来的空气声可以听到)。</li>
	<li>重复步骤3.5 和3.6。最后的苯酚溶液体积增加到样品是1毫升。</li>
	<li>重复步骤3.6 直到样品完全溶解。 注意:重要的是要释放所有的压力, 从管, 然后再继续, 以防止管爆裂。</li>
	<li>让样品在室温下站立, 同时重复步骤3.4 至3.8 为下一个样品。</li>
	<li>一旦所有样品被处理, 简要地漩涡在最大速度和孵育所有样品5分钟在室温。</li>
	<li>离心机10分钟在 12 000 x g 在4° c。把管子带回室温 注意:离心后, 3 阶段存在, 如图 2所示: 脂肪 (顶部), RNA (中) 和颗粒 (底部)。</li>
	<li>对于每个样品, 转移尽可能多的 RNA (中间粉红色层) 尽可能进入第一套相应的管与 P1000 吸管使用过滤尖端。更改示例之间的提示。</li>
	<li>为了确保由脂质 (顶层) 的 RNA (中间层) 的最小污染, 刺穿顶部与尖端附近的管侧。将 RNA 放入新的锥形管中, 而不接触新管的侧面, 以防止可能出现在尖端外部的脂质转移。</li>
	<li>将200µL 的纯氯仿添加到每个样品中, 并由十五年代的反转混合. 室温下孵育10分钟。</li>
	<li>离心机15分钟在 12 000 x g 在4° c 并且使管子回到室温。 注意:离心后, 3 阶段存在: RNA (顶部), 其中含有 DNA (中) 和红色酚-氯仿, 其中含有蛋白质 (底部) 的相间。</li>
	<li>对于每个样品, 转移尽可能多的含水的透明 RNA 包含相 (顶相) 的第二套相应的锥形管与 P1000 吸管使用过滤-提示。在每个示例之间更改提示。 注意:这种界面是脆弱的, 如果吸入的速度过快, 可能会污染顶部的阶段。作为替代方案, 使用 P200 吸管来减少干扰界面的几率。</li>
	<li>在每个样品中加入500µL 100% 异丙醇, 并通过反相混合 15 sec。</li>
	<li>室温孵育10分钟</li>
	<li>离心机10分钟在 12 000 x g 在4° c 并且使管子回到室温。通过倒置每根管子来丢弃异丙醇。 注意:一个小的白色 RNA 颗粒通常可以看到, 但有时可能不会。</li>
	<li>加入1毫升的75% 乙醇 (从100% 乙醇和无核糖核酸的水中制备) 到每个样品, 并在最大速度下短暂地涡流每管。</li>
	<li>离心机5分钟在 7 500 x g 在4° c 并且使管子回到室温。</li>
	<li>将每根管子倒置, 然后轻拍一张棕色的纸, 去掉剩下的75% 乙醇。 注意:如果需要, 使用过滤头, 以消除酒精附近的 RNA 颗粒。在示例之间更改提示。</li>
	<li>把盖子打开, 空气干燥的 RNA 颗粒约15-20 分钟。 注意:在这一步, RNA 小球可能变得半透明。</li>
	<li>由于 rna 的增溶从颗粒的大小依赖, 评估大小的颗粒, 以确定的体积核糖核酸无水将用于溶解的 rna。记下每个样本的音量 (10 到25µL)。 注意:这一步只是定性的。如果不能看到 rna 颗粒, 在样品中加入10µL 的无 rna 水。</li>
	<li>在每个样品中加入10-25 µL 的无核糖核酸水 (在步骤3.24 前确定的体积)。</li>
	<li>在60° c 的热块中孵育样品5分钟。简要地漩涡管子。</li>
	<li>在60° c 的同一个热块中孵育一个额外的5分钟样品。</li>
	<li>快速旋转所有的样品在一个微型旋转离心机和立即投入冰。</li>
	<li>确定 RNA 的浓度和纯度。执行反向转录 (RT), 以获得 cDNA 或存储在一个-80 ° c 冰箱样品, 以备日后使用。 注意:避免多次冻结/解冻周期, 因为这会降低 RNA 样本。</li>
</ol>4. 脂肪组织 RNA 浓度和纯度的测定
<ol><li>是否新鲜提取脂肪组织 RNA 样品, 溶解或预储存在一个-80 ° c 冰箱, 收集所有样本, 并允许他们解冻的冰, 直到程序开始。这应该在下一个步骤开始之前或隔离结束时进行, 以避免多次冻结/解冻循环。</li>
	<li>准备和识别两套无核糖核酸1.5 毫升锥形管, 并把它们放在一个管架。</li>
	<li>当所有的 RNA 样品都完全解冻后, 将样品涡旋1秒, 放回冰上。</li>
	<li>稀释你的 rna 100 倍在 1x te 解决方案中的第一套核糖核酸1.5 毫升锥形管使用过滤核糖核酸免费提示 (99 µL 1x te 溶液和1µL RNA 的管)。</li>
	<li>对每个示例重复此操作。 注意:根据所使用的试管, 每个分光光度计的体积可能不同。</li>
	<li>一旦所有样品被处理, 漩涡每个稀释的样品。</li>
	<li>按照分光光度计的协议, 在处理稀释的 RNA 样品之前使用空白 (1x TE) 来校准仪器。</li>
	<li>将稀释的 RNA 样品转移到石英试管, 并测定每个样品在 260 nm 的浓度。 注意:如果分光光度计不提供最终的 rna 浓度, 使用此公式计算: rna (µg/µL) = OD260 x 稀释因子 x (40 µg RNA/1 000 µL)。</li>
	<li>通过计算260/外径280的比率, 确定每个样本的 RNA 纯度。 注意:外径为260/外径280的比率在2.0 左右通常被视为纯为 RNA12。强烈建议对 RNA 质量进行验证。为了做到这一点, 把1µg 的 RNA 的1% 琼脂糖漂白凝胶制成的1x 的缓冲区 (89 毫米三碱, 89 毫米硼酸和2毫米 EDTA, pH 8.0) 和1% 漂白溶液13 (图 3)。非常好质量的 RNA 显示28S 和 18S rRNA 带和28S 带应该比18S 更加强烈。RNA 质量是可以接受的, 当两个 rRNA 波段是可见的, 在一个类似的强度。当18S 波段比28S 波段更剧烈时, 当涂片可见时, rna 质量就变得不足, 表明 rna 降解。</li>
	<li>为每个样品, 准备10µL 0.5 µg/µL 的 RNA 从股票在第二套核糖核酸1.5 毫升锥形管使用无核糖核酸水, 以稀释样品的需要。这些样品用于 RT 获得 cDNA。</li>
	<li>一旦所有样品被处理, 启动 RT 或存储在一个-80 ° c 冷冻库样品, 以备日后使用。</li>
</ol>5. 反向转录 (RT)
<ol><li>脂肪组织 RNA 样品是否刚被稀释到0.5 µg/µL 或预贮存在-80 ° c 的冷冻库中, 收集所有样品和试剂, 并允许它们在冰上解冻直至程序开始。</li>
	<li>将 PCR 管条架放在冰上。一旦足够冷准备和识别一组无核糖核酸 PCR 管条, 并把它们放在机架上。包括一个空白。</li>
	<li>为 RNA 的 dnasei 处理准备反应: 做一个主混合 (为所需的样品量 + 1 样品) 以下 (数量为1样品): 1 µL 10X 反应缓冲器与氯化镁2, 0.5 µL dnasei I (1 U/µL) 和7.5 µL 的核糖核酸-自由水使用过滤-提示。每管中分配9µL。</li>
	<li>添加1µL 0.5 µg/µL 的 RNA 从每个样本到相应的管, 把每条带子上的帽子和快速旋转所有的小条在一个迷你旋转离心机, 使样品到底部的管。为空白添加1µL 的无 RNA 水。</li>
	<li>在37° c 的热循环或加热块上孵育所有管条, 30 分钟, 将管条放回冰上的机架上。</li>
	<li>在每根管子上加1µL 的 EDTA (50 毫米), 在热循环或65° c 的加热块上孵育所有的管条, 10 分钟. 把管条放回冰上的架子上。</li>
	<li>做一个主混合 (为所需的样品数量加1样品) 以下 (数量为1样品): 1 µL 随机 hexamers (0.2 µg/µL) 和1µL 的 dNTP 混合 (包含每四 deoxynucleotides 的浓度 10 mM)。免除2µL 的主混合在每个管使用过滤提示。在示例之间更改提示。</li>
	<li>在65° c 的热循环或加热块上孵育所有管条, 5 分钟, 将管条放回冰上的机架上。</li>
	<li>做一个主混合 (为所需的样品数量加1样品) 以下 (数量为1样品): 4 µL 5x RT 缓冲器, 1 µL 核糖核酸抑制剂 (20 u/µL), 0.25 µL 反转录酶 (200 u/µL) 和1.75 µL 无核酸水。使用过滤后的小贴士, 在每管中免除7µL。在示例之间更改提示。</li>
	<li>通过设置以下程序在热循环中执行 RT:10 分钟在25° c, 30 分钟在50° c, 5 min 在85° c。把管子条放回冰上的架子上。</li>
	<li>准备并识别一组无核糖核酸的 PCR 管条, 并将其放在机架上。</li>
	<li>对于每个样品, 用超纯水将新管条中的1:5 稀释的 cDNA 的数量准备好。1:5 稀释的 cDNA 样本将用于实时 PCR。</li>
	<li>一旦所有样品被处理, 启动实时 PCR 或储存样品 (库存和稀释样品) 在一个-20 ° c 冰箱后使用。</li>
</ol>6. 脂肪组织中基因表达的实时 PCR
注意:避免将荧光染料暴露在光线下。下面的协议描述了 s1614的参考基因的放大。应根据感兴趣的基因对引物和 PCR 条件进行修改。
<ol><li>无论是新稀释的 cDNA 样本1:5 或预储存在一个-20 ° c 冰箱, 收集所有的样品和试剂, 让他们解冻的冰, 直到程序开始。</li>
	<li>在冰上放置实时 PCR 管条的机架。一旦足够冷准备和识别一组实时 PCR 管条, 并把它们放在机架上。准备每个样品和空白的一式两份。</li>
	<li>做一个主混合 (为所需的样品数量加1样品) 以下 (数量为1样品): 1.9 µL 的超纯水, 5 µL 的荧光染料 (如 SYBR 绿色) 和0.3 µL 的前引物和0.3 µL 的反向底漆。每管中分配7.5 µL。</li>
	<li>从每个样本中添加2.5 µL 1:5 稀释的 cDNA 到相应的管中。为毛坯添加2.5 µL 的超纯水。把帽子放在每个小条上。</li>
	<li>按照制造商的指示, 在实时 PCR 上设置以下程序:10 分钟在95° c 和40个周期十五年代在50° c, 三十年代在60° c, 三十年代在70° c。将样品带放入实时 PCR 机的转子, 启动程序。 注意:热循环条件可能因所使用的仪器和感兴趣的基因而异。mrna 表达的结果在图 3显示的瘦素 mrna 扩增的参考基因 S16 mrna 放大14,15。</li>
</ol>

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作者没有什么可透露的。

随着 HF 饮食喂养, 肥胖小鼠发现有增加的身体和白脂肪组织重量相比, 喂食一个 N 饮食小鼠。用苯酚溶液提取的 RNA 具有纯度好的样品。瘦素是脂肪主要由脂肪组织产生的, 并且与脂肪质量16呈正相关。正如所料, 瘦素 mRNA 表达增加的肥胖小鼠相伴与他们的脂肪质量。
此方法有几个关键步骤。主要一个是一个白色脂肪组织仓库的污染与另一个, 因为它是已知的?...

肥胖是一种世界性的流行病, 可能导致并发症, 如2型糖尿病1。饮食诱导的肥胖和转基因动物模型经常用于肥胖及其相关并发症的研究。传统上, 白脂肪组织被称为过剩能量的储存室, 主要由脂质组成, 而褐色脂肪组织将能量转化为热能2,3。脂肪组织是动态的, 并将扩大和收缩取决于许多因素, 如食物摄入量和体力活动。因此, 为了确定促成这些变化的因素, 需要有足够的脂肪组织收集和处理4。
在白色脂肪组织中, 一般认为皮下和内脏脂肪库有不同的性质, 如解剖定位和功能2,5。因此, 为了避免产生冲突的结果或大的变异性, 需要注意避免这些不同的脂肪库之间的交叉污染收集脂肪垫。
此外, 从小鼠白脂肪组织中分离 RNA 或蛋白质有三大挑战。首先, 在肥胖小鼠身上收集脂肪垫并不是一件容易的事, 因为与其他器官如肾脏和心脏6相比, 不同的白色脂肪库的边界并不总是清晰的。其次, 由于脂肪组织的高脂质含量, 在 RNA 或蛋白质分离期间, 一层油脂漂浮在顶部, 防止直接进入样品。第三, 相对于褐色脂肪组织或其他组织, 白脂肪组织有相当低的 RNA 和蛋白质含量, 这是主要的关注, 当使用小鼠, 喂食正常 (N) 饮食和小鼠, 预计将有低脂肪质量 (即KO 模型, 药物治疗, 运动训练等)7,8。
因此, 选择适当的方法分离 RNA 从脂肪组织是关键的。苯酚/氯仿萃取的替代方法是商业套件。它们通常基于最初的苯酚提取步骤, 然后在一列9上进行 RNA 纯化。然而, 这些套件通常更昂贵, 并提供较低的产量样本, 而 RNA 质量可能是可变的, 但更少的时间消耗。然而, 这里所描述的苯酚溶液/氯仿萃取的最大优点之一是可能从单个样本10中分离出 RNA、DNA 和蛋白质。由于小鼠脂肪垫通常是小的, 并给出少量的 RNA 和蛋白质 (特别是在精益小鼠模型), 这些协议最大化的数据, 你可以得到一个小样本。
本文的目的是详细描述一个方法, 以确保足够的样本收集从三小鼠白脂肪组织仓库, 以及数量和质量的 RNA 隔离。通过遵循该协议获得的 RNA 可用于实时 PCR 检测。该协议不是为了 RNA 分离培养的原发脂肪细胞。

<table><tbody><tr><td>1 mL seringes</td><td></td><td></td><td></td></tr><tr><td>1X TE solution (10 mM Tris-HCl and 1 mM EDTA&amp;bull;Na&lt;sub&gt;2&lt;/sub&gt;. pH 8.0)</td><td></td><td></td><td></td></tr><tr><td>22 G needles</td><td></td><td></td><td></td></tr><tr><td>26 G needles</td><td></td><td></td><td></td></tr><tr><td>75% Ethanol</td><td></td><td></td><td></td></tr><tr><td>Block heater (dry bath)</td><td></td><td></td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C2432-500mL</td><td></td></tr><tr><td>dATP&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>Thermo scientific</td><td>R0141</td><td></td></tr><tr><td>dCTP&amp;nbsp;&amp;nbsp;</td><td>Thermo scientific</td><td>R0151</td><td></td></tr><tr><td>dGTP&amp;nbsp;&amp;nbsp;</td><td>Thermo scientific</td><td>R0161</td><td></td></tr><tr><td>DNase I (1 U/&amp;micro;l)&amp;nbsp;</td><td>Thermo scientific</td><td>EN0521</td><td></td></tr><tr><td>dTTP&amp;nbsp;</td><td>Thermo scientific</td><td>R0171</td><td></td></tr><tr><td>Faststart Universal SYBR green Master (Rox)</td><td>Roche</td><td>4913922001</td><td></td></tr><tr><td>Faststart universal SYBR green master (Rox) fluorescent dye&amp;nbsp;</td><td>Roche</td><td>4913914001</td><td></td></tr><tr><td>Filtered tips</td><td></td><td></td><td></td></tr><tr><td>Forceps&amp;nbsp;</td><td>Instrumentarium</td><td>HB275</td><td></td></tr><tr><td>Gauze</td><td></td><td></td><td></td></tr><tr><td>Hammer</td><td></td><td></td><td></td></tr><tr><td>High fat rodent diet</td><td>Bio-Serv, Frenchtown, NJ</td><td>F3282</td><td></td></tr><tr><td>Isopropanol&amp;nbsp;</td><td>Laboratoire MAT</td><td>IH-0101</td><td></td></tr><tr><td>Leptin forward PCR primer (5&amp;rsquo;-GGGCTTCACCCCATTCTGA-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>Leptin reverse PCR primer (5&amp;rsquo;-GGCTATCTGCAGCACATTTTG-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>Liquid nitrogen</td><td></td><td></td><td></td></tr><tr><td>Maxima Reverse Transcriptase (enzyme and 5x buffer)</td><td>Thermo scientific</td><td>EP0742</td><td></td></tr><tr><td>Nanopure water (referred as ultrapure water)</td><td></td><td></td><td></td></tr><tr><td>Nitrile examination gloves</td><td></td><td></td><td></td></tr><tr><td>Nitrile gloves</td><td></td><td></td><td></td></tr><tr><td>Normal rodent diet</td><td>Harlan Laboratories, Madison, WI</td><td>Harlan 2018</td><td></td></tr><tr><td>P1000 pipetman</td><td></td><td></td><td></td></tr><tr><td>P2 pipetman</td><td></td><td></td><td></td></tr><tr><td>P20 pipetman</td><td></td><td></td><td></td></tr><tr><td>P200 pipetman</td><td></td><td></td><td></td></tr><tr><td>Phenol solution (TRIzol)&amp;nbsp;</td><td>Ambion Life Technologies</td><td>15598018</td><td></td></tr><tr><td>Pre-identified aluminium foil</td><td></td><td></td><td></td></tr><tr><td>Quartz spectrophotometer cuvette</td><td></td><td></td><td></td></tr><tr><td>Rack for PCR tube strips</td><td></td><td></td><td></td></tr><tr><td>Racks for RT-PCR tube strips</td><td></td><td></td><td></td></tr><tr><td>Random hexamers&amp;nbsp;</td><td>Invitrogen</td><td>58875</td><td></td></tr><tr><td>Real-time PCR Rotor Gene system&amp;nbsp;</td><td>Corbett research</td><td>RG-3000 Rotor-Gene thermal cycler</td><td></td></tr><tr><td>Refrigerated bench-top centrifuge</td><td></td><td></td><td></td></tr><tr><td>RiboLock RNase Inhibitor&amp;nbsp;</td><td>Thermo scientific</td><td>EO0381</td><td></td></tr><tr><td>RNase-free 1.5 mL eppendorf tubes</td><td></td><td></td><td></td></tr><tr><td>RNase-free 1.5 mL screw cap tubes</td><td></td><td></td><td></td></tr><tr><td>RNase-free PCR tube strips (0.2 mL) and caps</td><td></td><td></td><td></td></tr><tr><td>RNase-free water&amp;nbsp;</td><td>Hyclone</td><td>SH30538.02</td><td></td></tr><tr><td>RT-PCR machine</td><td>Qiagen</td><td>Rotor-Gene Corbett 3000</td><td></td></tr><tr><td>RT-PCR tube strips (0.1 mL) and caps</td><td></td><td></td><td></td></tr><tr><td>S16 forward PCR primer (5&amp;rsquo;-ATCTCAAAGGCCCTGGTAGC-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>S16 reverse PCR primer (5&amp;rsquo; ACAAAGGTAAACCCCGATCG-3&amp;rsquo;) 10 uM</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer</td><td>Biochrom</td><td>Ultrospec 3100 pro</td><td></td></tr><tr><td>Stainless steel mortar and pestle</td><td></td><td></td><td></td></tr><tr><td>Surgical pads</td><td>Home made a foam board wrapped in a disposable absorbent underpad</td><td></td><td></td></tr><tr><td>Surgical scissors&amp;nbsp;</td><td>Intrumentarium</td><td>130.450.11</td><td></td></tr><tr><td>Thermal cycler</td><td></td><td></td><td></td></tr><tr><td>Thermal cycler&amp;nbsp;</td><td>Biometra</td><td>Thermocycler</td><td></td></tr><tr><td>Vortex mixer</td><td></td><td></td><td></td></tr><tr><td>Weighing spatula</td><td></td><td></td><td></td></tr></tbody></table>

mouse adipose tissue collection and processing for rna analysis

与其他组织相比, 白脂肪组织的 RNA 和蛋白质含量在诸如实时 PCR 和西方印迹等下游应用中有相当少的成分, 因为它主要含有脂类。RNA 分离从脂肪组织样本也有挑战性, 因为需要额外的步骤, 以避免这些脂质。在这里, 我们提出了一个程序收集三从小鼠解剖不同的白色脂肪组织, 以处理这些样本和执行 RNA 隔离。我们进一步描述了用实时 PCR 技术合成 cDNA 和基因表达实验。本协议允许减少动物的头发和血液对脂肪垫的污染, 以及在组织收集期间不同的脂肪垫之间的交叉污染。它也被优化, 以确保足够数量和质量的 RNA 提取。该协议可广泛应用于任何小鼠模型, 其中脂肪组织样本需要进行常规实验, 如实时 PCR, 但不打算与原发脂肪细胞培养分离。

按照尸检程序, 三白脂肪组织收集和加权两组小鼠 (N 和 HF 饮食喂养小鼠)。正如预期的那样, HF 饮食中的小鼠在 N 饮食 (表 1) 上与窝相比, 增加了最终体重和体重增长。这些观察伴随着超过2倍的体重增加的 PGF, 重复频率和超重的肥胖小鼠相比, 在 N 饮食。
在对分离的 RNA 进行任何实验之前, 它的纯度被评估如步骤4.9 ?...

Watch this Scientific Journal Video about Mouse Adipose Tissue Collection and Processing for RNA Analysis at JoVE.com

Mouse Adipose Tissue Collection and Processing for RNA Analysis

小鼠脂肪组织的采集和 RNA 分析处理

本文的目的是提出一步一步的程序, 收集不同的白脂肪组织的小鼠, 处理脂肪样本和提取 RNA。

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

mouse-adipose-tissue-collection-and-processing-for-rna-analysis

Research

JoVE Journal

Biochemistry

48.2K 次观看.  Université de Montréal. 本文的目的是提出一步一步的程序, 收集不同的白脂肪组织的小鼠, 处理脂肪样本和提取 RNA。

视频: Mouse Adipose Tissue Collection and Processing for RNA Analysis

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

科研

神经科学

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

单细胞基因组应用的脂肪组织核分离

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

遗传学

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of arthritis research, many studies are focused on inflamed joints that are composed of a complex mixture of bone, cartilage, muscle, stromal cells and immune cells. Here, we established a reliable and robust mechanical method to disrupt inflamed mouse paws into homogeneous pulverized samples in a cryogenically controlled environment. Protein and RNA lysates were processed to enable proteomic and transcriptional endpoints and molecular characterization of relevant disease pathways in local tissue.

A cryogenic pulverization method to process murine paws using a liquid nitrogen freezer mill was developed to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses.

在胶原蛋白诱发关节炎模型中，用于处理小鼠爪的冷冻粉碎协议，用于评估组织炎症的分子通路

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of ...

免疫与感染

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

用于下游分析的冷冻啮齿动物大脑区域集合

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

从小鼠胰腺RNA提取:核糖核酸酶丰富的组织

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

生物学

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

医学

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

从小鼠眼中激光捕捉高纯大梁网的基因表达分析

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...