This research was supported by a team grant from Movember/Prostate Cancer Canada to JMSB, DMB, PCP, and JL, and by the Ontario Institute of Cancer Research (JMSB, DMB, and PCP) and Motorcycle Ride for Dad Kingston/University Hospitals Kingston Foundation/Kingston General Hospital (DMB, PCP).

1.组织取心<ol><li>审查显微镜载玻片并使用细点永久性标记勾画感兴趣区域（S）。切出石蜡膜足以覆盖的在显微镜载玻片感兴趣区域的一个部分。固定放在反转片和缠绕膜的边缘上，以保持薄膜打滑。使用细点永久性标记，轮廓的整个组织和在组织内感兴趣区域（S），保持轮廓接触 - 但以外 - 该（多个）区域。 </li><li>除去薄膜，并将其传输到相应的组织块。通过翻转或旋转它，使整个组织的轮廓块中的组织的观察形状相匹配（ 图1）定向膜。按薄膜的部分牢牢到块以防止滑动的表面上。 </li><li>使用永久性标记的一角，使浅但可见（大约需要0.2 MM）压痕沿线地区（S）的大纲terest，然后取出薄膜。负载1毫升漂白剂，70％的乙醇和水的成单独的1.5或2.0 ml微量离心管。 </li><li>清洗从0.6mm的冲头通过在尖端浸入含有管漂白滑动冲上下数次设置受体（红色）打孔。重复用70％乙醇，然后用水将上述步骤（临界确保漂白被移除）。 </li><li>按下冲头进入组织，感兴趣的区域内，以3毫米的深度和撤回冲头。松开核心进入低推它用手写笔打孔装订1.5或2 ml管。储存在-20℃（长期）或4℃下短期使用的内核。 </li><li>清洁按照步骤1.4和继续下一个区域或样品一拳。 </li></ol> 2. Deparaffinize的FFPE组织核心<ol><li>通过加入1毫升二甲苯到组织核心和10秒剧烈振荡在1.5或2 mL管结转脱石蜡。 HEA吨在50℃下3分钟。 </li><li>离心机在室温（RT）和最大速度（21130 XG）并置于管在冰上5分钟，2分钟（允许蜡状残余物以固化在上面）。 </li><li>小心取出使用周围枪头与上清半月板积累了石蜡和重复治疗二甲苯（步骤2.1-2.2）。 </li><li>加入1毫升乙醇（100％）和涡流剧烈持续10秒。离心在RT（最大速度）2分钟，然后小心地弃去乙醇。重复上述步骤一次。 </li></ol> 3.脱蜡核同质化<ol><li>重悬在700微升的乙醇（100％）之前均匀化的核心。使用电动组织匀浆，碾核成细小颗粒组织（约1分钟中等设置）。清洗各样品之间的匀化器探针，以尽量减少结转污染。 <ol><li>填充15ml试管以〜10毫升漂白剂，核糖核酸酶和溶液和70％的乙醇。样本同质后genization，洗匀化器探针中的每个在上述的顺序清洗液的。在清洗阶段运行的最高速度均质。 </li><li>擦拭用纸巾探头并让探头均质下一个样品之前完全干燥。目视检查探测叶片残留组织碎片。如果找到，再次清洁探头。每日更换该清洁溶液（漂白剂，乙醇和核糖核酸酶和溶液）。 </li></ol></li><li>以下均化，通过添加更多的100％乙醇（〜300微升）使样品体积至1ml。离心最大速度为15分钟，用RNA提取前仔细吸出约15-20分钟的乙醇和空气干燥颗粒。 </li></ol> 4.消解蛋白酶K <ol><li>重悬150微升蛋白酶K消化缓冲液沉淀和甩尾管松动沉淀。加10μl温度稳定的蛋白酶K，并通过轻弹混匀（不要涡管）。孵育在管中的内容在56℃适度搅拌15分钟。 </li><li>允许管在冰上孵育3分钟。完全冷却是在下列步骤有效沉淀重要。离心以最大速度15分钟。 </li></ol> 5.从DNA分离RNA <ol><li>小心转移上清液，而不会干扰沉淀，至新的1.5毫升RNA纯化。 </li><li>保持用于DNA纯化粒料（粒料可以在RT在-20℃储存2小时，在2-8℃下1天，或者更长的时间）。 </li></ol> 6. RNA纯化<ol><li>孵育在80℃下进行15分钟的含有RNA的上清液（不超过这个时间）。接着，短暂离心所述管，以从盖的内侧收集液滴。 </li><li>加入320微升缓冲液RLT调整结合条件，并通过吹打混合。接下来，添加720微升乙醇（100％），和旋涡。 </li><li> 6转让00微升的样本，包括可能已经形成，放置在2 ml收集管RNA离心柱（试剂盒提供），并预留剩余内容的任何沉淀。离心机在≥8,000XG 15秒，弃去流通和再利用的收集管。 </li><li>转印剩余的样品上柱，包括可能在管的盖已经积累的液滴，离心15秒在≥8,000XG，并弃去流过。 </li><li>添加350微升缓冲液FRN的旋转柱和离心机15秒在≥8,000XG，丢弃流过和重用收集管。 </li><li>轻轻混匀10微升DNA酶I原液用70微升缓冲液RDD，直接加入到离心柱膜，并在室温孵育15分钟。 </li><li>加入500μlBuffer FRN的旋转柱，离心15秒在≥8,000XG和保存流通用于下一步骤中使用。为了提高小RNA的复苏，将离心柱中新2 ml收集管和来自前面步骤的旋转柱应用流通。 </li><li>离心机在≥8,000XG 15秒，弃去流通和再利用在下一步骤中，收集管。加入500μl缓冲液RPE到离心柱并离心15秒在≥8,000XG，丢弃流通和再利用在下一步收集管。 </li><li>加入500μl缓冲液RPE到离心柱并离心15秒在≥8,000XG和丢弃流过的收集管。 </li><li>放置旋转柱在新的2ml收集管中，打开盖子和离心机以最大速度为5分钟。丢弃流过的收集管。 </li><li>放置旋转柱在一个新的1.5 ml收集，加入20微升的无RNase水直接在旋转柱膜，孵育所述管在室温下1分钟。离心机以最大速度为1分钟以洗脱RNA。储存在-80℃下洗脱的RNA样品。 </li></ol><ol><li>悬浮通过逐步加入45微升蛋白酶K缓冲的RNA提取过程中获得的丸粒（400毫摩尔Tris 7.5，400 mM氯化钠，3 mM的氯化镁 ，4％SDS）; 45微升H 2 O;和400微克的高效力蛋白酶K的</li><li>在56℃下放置24小时孵育上述溶液（推荐）或过夜。 Performincubation在90℃2小时，无需搅拌，短暂离心所述微量离心管，以从所述盖的内收集液滴。 </li><li>使样品冷却至室温，然后加入4微升RNA酶A（100毫克/毫升）。在室温下孵育2分钟的样品。 </li><li>加入200μl缓冲液AL到样品，并通过涡旋调匀。接着，加入200微升的100％乙醇，并通过涡旋调匀。在≥8,000×g下将整个样品转移到提供旋转柱，放置在2 ml收集管中，并离心1分钟。 </li><li>放弃与收集管流通，并将旋转柱在新的2ml收集管中。加入700μl缓冲液AW1至旋转柱，离心15秒在≥8,000XG，丢弃流通和再利用的收集管。 </li><li>加入700μl缓冲液AW2的旋转柱，离心15秒在≥8,000XG，丢弃流通和再利用的收集管。接着，在≥8,000XG加入700微升的100％乙醇，以旋转柱，离心15秒，并丢弃与流通的收集管。 </li><li>放置旋转柱在新的2ml收集管中，打开该旋转柱的盖，和离心机全速5分钟。丢弃流过的收集管。 </li><li>放置旋转柱在一个新的1.5 ml收集管，并添加25微升加热无核酸酶水（50℃）。在50℃下孵育柱和管10分钟。离心机以最大速度1分钟，加入25μl的不含核酸酶的水（RT）的柱，并孵育在RT 1分钟。 </li><li>离心机以最大速度（21130 XG）和含有基因组DNA（脱氧核糖核酸约总计50μl）收获流过1分钟。在-20℃下储存列（如果另一洗脱后需要）。 </li></ol>

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The authors declare no competing financial interests.

用于从感兴趣的组织区域的DNA和RNA的成功提取，准确核化是至关重要的。这个协议描述了使用组织冲床以分离0.6毫米直径铁心和概述用于从显微镜传输符号滑动以对应FFPET块的过程。进行必要的修改，以生产商的方案，以有效地提取从内核核酸，这是大约比的量，协议是预期的切片机切片厚50倍。由于芯可以包含相对更石蜡组织切片，通过反复二甲苯和乙醇处理步骤芯有效脱蜡被要求。的脱蜡后步骤的成功取决于适当的机械组织核心的同质化和高效的蛋白酶K消化。能够进行蛋白酶K消化的进一步优化。 值得一提的是，此方法鉴定块的表面上的兴趣外资企业领域，如在对应的组织病理学载玻片识别。由于核心收成组织，可能是深3或4毫米，这个协议的用户可能会关注什么细胞或组织奠定了块表面之下。虽然这是一个有效的关注，多个研究（参照27中综述）已证明组织核心忠实代表病理组织块的组织学和分子的特性，尤其是当重复或三次重复芯从感兴趣的区域进行采样。 如在该协议通过的修改商业提取试剂盒使DNA和RNA从相同的组织的并发萃取，协议节省了宝贵的生物材料，并允许从同一样品在两个所得核酸之间的直接比较。 RNA和DNA的提取同期减少了劳动力和组织消耗了一半，并使基因expr的精确综合分析分裂国家，以及在DNA中发现的表观遗传和基因的功能。因为RNA和DNA从这些代表性组织核心的产率通常超过分别600和300毫微克，并且由于大多数当前PCR和下一代测序应用通常需要10-100纳克，大部分由该协议纯化样品应提供足够的材质为几个下游检测。此协议已被证明是在整个独立的实验室可再现（Selvarajah 等人 ， 在准备）。这个协议的RNA是足够的质量，使用任一RT-PCR或一个流行多分析平台的基因表达分析，及DNA的甲基化特异性PCR测定表现良好。旨在评估回收的核酸下一代测序工具未来的研究是必要的。 因此，若干修改对市售的协议，设计为薄FFPET部分制成，使得它适合初学者r处的RNA和DNA的从0.6毫米FFPET芯共萃取。该协议证明了大队列的前列腺癌样本和在从乳房，脑，膀胱癌有限的一组样品的一致的高产量。总体而言，该协议应允许用户进行大型注释良好的组织聚集针对性基因为基础的分析。重要的是，该协议允许在FFPET感兴趣区域的有效集中的采样的，相对小动手时，对于大多数下游应用足够高的产率。

基因组生物标志物的研究旨在确定分子相关因素能够准确和可靠地反映疾病状况，并且在临床上有用的方式这样做。1生物标志物的开发是在良好的注释组织样品的回顾性分析依赖。患病的和正常组织样品储存或者作为新鲜冷冻组织专门生物库或作为临床档案福尔马林固定石蜡包埋的组织（FFPET）块。新鲜冷冻组织允许高质量的核酸提取，并已广泛地应用于基因组生物标志物发现的研究。2,3然而，更少的组织样品中的生物库可用，并研究这种组织朝较大的样品，不寻常的类别引入了偏压病，并且在以更大的能力，以银行组织专业中心看到患者的4 FFPET，相反，是用于患病的人及动物组织的默认存储方法。虽然FFPET块维持细胞米orphology，固定过程交联的其他细胞成分的核酸。交联的RNA和DNA是可恢复的，但只在退化，高度分散的形式。5,6-然而，这些DNA和RNA片段适合于分析由扩阵列测定法，包括基因表达，DNA的甲基化和有针对性的测序。 7,8-要利用这个机会在大数量和品种可用于研究FFPET的，有需要一种有效和可靠的提取方案。 生物标志物研究的组织有很大一部分集中于癌症。像其他类型的病变组织，癌组织往往显示了在细胞保存和细胞类型显著区域异质性。因为生物标记研究依赖于与分子功能病变组织的组分相关的能力，此过程中的关键步骤是组织的精确收获是保存完好，并富集对于disease正在研究中。在FFPET两个富集技术通常用于:激光捕获显微切割（LCM），和切片机切片。 LCM能够实现高度集中的组织收获，可用于在异构组织中分离出特定的，保存完好的细胞类型9,10然而，LCM需要昂贵的设备，是过于耗时大量样品。切片机切片是一种更广泛使用的过程，其中薄片是从FFPET块切11,12切片机切段通常包括组织是在细胞保存异构（ 例如 ，坏死与保存完好）和成分（ 如癌症与良性实质），因此可能导致的单独研究分子的特性最好的同质化。因此，存在需要一种丰富了感兴趣的细胞的高通量方法。第三种方法中，从FFPET芯核酸的分离，提供了这种富集，是适合于高通过量hput协议，并已被他人使用分离从分离组织芯的RNA或DNA。7,13,14 许多出版的协议指定从FFPET（ 表1）中提取核酸的方法。然而，在RNA和DNA是从相同的组织中提取的协议已经被用于切片的组织切片进行了优化，但不用于组织芯。15,16同样，公布的协议而提供更高的组织特异性，或者通过组织芯或滑动microdissections，指定程序为提取DNA，但不RNA。7,17这里，对于DNA和RNA从相同的组织核心的双提取一个优化的协议是证明。组织核心是通过插入组织微阵列（TMA）成拳映射到FFPET块地区的利益收获。映射由注释用记号笔显微镜载玻片和注释传送到相应的FFPE的表面进行Ť块（ 图1）。 在此之前的工作，促成该协议的发展包括一些市售的核酸提取系统的比较。在这种比较，修改商业协议，如下所述提供最高的DNA和RNA的产率和质量（Selvarajah 等人 ， 在准备 ）。组织核心比通常FFPET提取方案11,12,14,18用的5-10微米的微米切片厚- 20，并且可以包含更多的不同量石蜡。为了弥补这一点，脱蜡通过重复二甲苯和乙醇处理，并通过引入电动均化步骤（ 图1）提高。此外，蛋白酶K消化时间被延长以增加DNA产量。总体来说，这个协议是符合成本效益，使在La疾病的分子和组织病理学特征之间建立联系RGE，以及表征的人群。其全部的协议能够可靠2天内进行，其中包括3小时动手时，很少需要专门的或昂贵的设备。 在一步一步协议是以下简称制造商的方案的一个修改的版本。21请参见材料/设备的表针对特定试剂，设备和制造商。

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Plastic paraffin film, &#34;Parafilm &#39;M&#39;&#34;</td>
			<td style="width:217px;">Bemis&#160;</td>
			<td style="width:119px;">RK-06720-40</td>
			<td style="width:167px;">Any generic paraffin film will work as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Sodium Hypochlorite, &#34;Ultra Bleach&#34;</td>
			<td style="width:217px;">Likewise</td>
			<td style="width:119px;">53-2879-2</td>
			<td>Any generic bleach will work as a substitute. Hazardous material that can cause burns on contact.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Molecular biology grade absolute ethanol</td>
			<td style="width:217px;">Fisher BioReagents</td>
			<td style="width:119px;">BP2818-500</td>
			<td>Sigma-Aldrich E7023 suffices as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Molecular Grade H2O</td>
			<td style="width:217px;">G-Biosciences</td>
			<td style="width:119px;">786-293</td>
			<td>Sigma W4502 suffices, as well as any other brand of molecular grade H2O</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">0.6mm Punch Set for Beecher Instruments&#160;</td>
			<td style="width:217px;">Estigen</td>
			<td style="width:119px;">MPO6[Yellow]</td>
			<td>Make sure to use the red receiver punch from the set</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Fine point permanent marker</td>
			<td style="width:217px;">Sharpie</td>
			<td style="width:119px;">10365796S</td>
			<td>Using the marker on FFPE tissues causes it to dry out quickly, so several may be required</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">FFPE tissue block</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Stained tissue slide corresponding to FFPE block</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">1.5 mL Micro-Centrifuge Tubes</td>
			<td style="width:217px;">Fisher BioReagents</td>
			<td style="width:119px;">05-408-137</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">2.0 mL Low binding tubes (LoBind Micro-Centrifuge Tubes)</td>
			<td style="width:217px;">Eppendorf</td>
			<td style="width:119px;">22431048</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">1.5 mL Low binding tubes (LoBind Micro-Centrifuge Tubes)</td>
			<td style="width:217px;">Eppendorf</td>
			<td style="width:119px;">22431021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Histology Xylene</td>
			<td style="width:217px;">VWR</td>
			<td style="width:119px;">CA 95057-822</td>
			<td>Fisher Scientific X5-500 suffices as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Molecular Biology Grade 2-Propanol</td>
			<td style="width:217px;">Sigma</td>
			<td style="width:119px;">I9516</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:283px;">AllPrep FFPE DNA/RNA Kit&#160;</td>
			<td style="width:217px;">Qiagen</td>
			<td style="width:119px;">80234</td>
			<td>Prepare buffers accodring to the AllPrep DNA/RNA FFPE Handbook21</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Buffers: RLT, FRN, RPE, ATL, AL, AW1, AW2, DNaseI solution</td>
			<td style="width:217px;">Qiagen</td>
			<td style="width:119px;">80234</td>
			<td>Prepare buffers accodring to the AllPrep DNA/RNA FFPE Handbook21</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Temperature stable proteinase K</td>
			<td style="width:217px;">Qiagen</td>
			<td style="width:119px;">80234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">High potency proteinase K</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:119px;">25530-049</td>
			<td>Invitrogen 25530-015 suffices as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAse neutralizing solution (Rnase AWAY)</td>
			<td style="width:217px;">Molecular BioProducts</td>
			<td style="width:119px;">7003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">RNaseA 100mg/mL</td>
			<td style="width:217px;">Qiagen</td>
			<td style="width:119px;">19101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">BD Integra Syringe 3mL 21G x 1/2</td>
			<td style="width:217px;">BD&#160;</td>
			<td style="width:119px;">305274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Motorized tissue homogenizer (TissueRuptor)</td>
			<td style="width:217px;">Qiagen&#160;</td>
			<td style="width:119px;">9001271</td>
			<td>Fisher Scientific 14-261-29 suffices as a substitute</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">-20&#176;C and -80&#176;C Laboratory Freezer &#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Micro-Centrifuge with rotor for 2mL tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Digital Vortex Mixer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Pipettes and filter tips</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Heating blocks or water baths</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Tris Hydrochloride&#160;</td>
			<td style="width:217px;">Amresco</td>
			<td style="width:119px;">0234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Sodium Chloride&#160;</td>
			<td style="width:217px;">Amresco</td>
			<td style="width:119px;">0241</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Anhydrous Magnesium Chloride</td>
			<td style="width:217px;">Sigma</td>
			<td style="width:119px;">M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Sodium Dodecyl Sulfate</td>
			<td style="width:217px;">Sigma</td>
			<td style="width:119px;">L4509</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Acrodisc 25mm syring filters with 0.45 um Supor membrane</td>
			<td style="width:217px;">Pall</td>
			<td style="width:119px;">PN 4614</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:283px;">Syringe with retracting BD PrecisionGlide needle 3mL</td>
			<td style="width:217px;">BD Integra</td>
			<td style="width:119px;">305274</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:283px;">Hydrochloric Acid</td>
			<td style="width:217px;">BDH</td>
			<td style="width:119px;">3026</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Multianalyte gene expression platfrom (nCounter &#174; CAE codeset and Nanostring nCounter platform)</td>
			<td style="width:217px;">Nanostring nCounter platform, Nanostring</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:283px;">Fluorometric nucleic acid quantification (Qubit dsDNA HS Assay Kit and Qubit&#174; RNA BR Assay Kit)</td>
			<td style="width:217px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of formalin-fixed paraffin-embedded tissue cores for both rna and dna extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

该协议代表用于从组织核心中回收的DNA和RNA，利用设计用于组织切片商用提取系统的变型的优化方法。优化包括引入组织均质化的，更有效的蛋白酶K的利用DNA提取，以及组织消化时间延长。图表和统计分析包括2路方差分析，线性回归和相关性。 蛋白酶消化的优化 的商品试剂盒包括其取代有一个更有效的蛋白酶K，导致更高的DNA产量（ 图2A）室温下稳定的蛋白酶K溶液。为了进一步提高该DNA产率，消化被延长为2至24小时。无显著差异所看到的两个时间点之间，但24小时消化似乎提供跨越山更加一致的产量mples。然而，进一步温育48小时并没有进一步提高DNA回收率（ 图2B; P = 0.74）。 从FFPE前列腺癌组织样品典型DNA回收使用优化的协议，RNA和DNA进行共萃取从333前列腺癌FFPET样品从3至14岁的在样品的年龄。从每个样品中，3个组织核心（0.95±0.13 mm 3的平均总的组织体积）用作输入。虽然有其他微流体根据其可以估算浓度和提供的核酸分子的大小分布的评价凝胶电泳方法中，这样的方法不提供可重复的核酸定量，以及RNA和DNA之间不能区分作为flourometrically基于分析做。 22，因为基于微凝胶电泳结果是不可靠的支离破碎从FFPET来源的核酸，进行荧光测定测量23核酸收益率（详见试剂列表）。平均收益率为RNA 2270纳克和820纳克DNA（ 图3A）的。在这项研究中分析的所有样品FFPET大约90％的DNA产生的≥100纳克和RNA≥500纳克。有趣的是，在FFPET样品和核酸的回收（ 图3B）的年龄之间没有相关性显著。总体而言，RNA和DNA产率跨越样本相关（R 2 = 0.39; P &lt;0.0001），但如超过两倍多的RNA比DNA从各样品（ 图3C）回收。 为先导，优化工作于前列腺组织进行的，下一步是调查这个协议对几个附加类型的存档组织的性能。开始用手术切除及尸检样品FFPET是代表nign肝（1样品1的情况下），脑（从1例8个样品），膀胱（从2情况2样品），和乳腺（从3 3,3样本）的癌症，该协议得到的DNA&gt; 100毫微克和RNA从90％的样品（ 图3D）。而核酸产率在尸检组织中比在手术组织下，有代表性的结果表明，该协议两端产生从不同的站点来源的癌症类似的产量。 和DNA RNA的评估完整性和下游分析他们的代表性能使用为FFPET优化的商业多分析基因表达的平台进行的47个基因中的8选FFPET前列腺癌样本和一个新鲜PC-3前列腺癌细胞系样品（作为阳性对照）RNA表达分析。在PC3中mRNA的计数均通常比那些从FFPET山更高mples（ 图4A）。然而，在比较所有基因的相对表达，FFPET前列腺癌样品显示类似的表达谱与PC-3的RNA，这表明RNA的两种来源都适合的RNA表达谱。 为了阐明用此协议中提取的基因组DNA的性能，从FFPET样品亚硫酸氢盐转化的DNA提取物通过甲基化扩增特异性PCR（MSP）。24 ALU重复元件，高度甲基化区域中存在的数百万份中的人类基因组的MSP分析， 25是作为基因组的甲基化的控制，并且预计将显示样品之间的最小的变化。如图4B所示，有很少或几乎没有在ALU MSP甲基化水平不同样品之间看到变异。此外，基于GSTP1，已知在前列腺癌，但是良性样品没有被甲基化的基因的MSP分析中，26显示没有可检测的amplifications从良性样本的DNA。正如预期的那样，在从癌组织中的DNA中检测到较低的qPCR周期阈值，这表明甲基化的GSTP1份数富集。核酸本协议回收的实用程序典型的下游测定中进一步测试，使用核酸良性肝和脑（验尸）回收并从两个手术切除乳腺癌样本。无论RT-qPCR的基于表达式和MSP检测乳腺癌和肝癌FFPET表现不错，但RT-PCR检测未能放大来自验尸脑肿瘤样品中高度表达的mRNA（ 图4C），这表明RNA已降解，可能是由于延迟组织固定。 <img alt="图1" src="/files/ftp_upload/54299/54299fig1.jpg" /> 图 1: 为FFPET样品提取步骤概述下图说明了在利益如何面积组织块是基于从显微镜载玻片组织病理学选择映射。三0.6毫米组织核心，然后从各个组织区域使用活检拳，一起均化，然后进行RNA和DNA提取得到的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54299/54299fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54299/54299fig2.jpg" /> 图 2: 核酸（DNA和RNA）的毫微克/ 从两个蛋白酶K（高温度稳定性和高效力酶），并在孵化时间为后者的范围内测试 （A）蛋白酶K的性能收益 FFPET 毫米 3从不同的供应商。 DNA提取用配合K提供的温度稳定酶有代表性的FFPET样品进行它与其他制造商更有效的酶。 （b）确定最佳蛋白酶K孵育时间最大化DNA产量。高浓度的蛋白酶K消化的性能在使用3 FFPET样本三种不同的培养时间进行了评估。误差棒代表平均值（SEM）的标准误差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54299/54299fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54299/54299fig3.jpg" /> 图 3: 核酸（DNA和RNA）以ng FFPET的 /毫米 3 的合计，横跨样本年份和代表组织类型 产率 （A）总回收从福尔马林固定的石蜡包埋的组织的核酸。提出的核酸量是基于333 FFPET样品的提取ù唱优化的协议。 （B）中回收的总DNA和RNA和FFPET样品年龄的关系图。所用的萃取FFPET样品从2000年从333前列腺样品同时提取的DNA和RNA产量之间获得至2012（C）的关系。有DNA和RNA产量之间的正相关性。使用额外的存档组织类型的协议（D）示范。优化的协议被用于从14的癌症（乳腺癌，膀胱癌和脑）和正常（肝脏）的样品中提取核酸。误差棒代表SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54299/54299fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54299/54299fig4.jpg" /> 图4: 从Tissu酒店RNA和DNA的性能联合萃取在下游应用E型磁芯。（A）的mRNA算个FFPE前列腺癌组织和新的PC-3细胞系的控制。每个点代表分别萃取3技术重复的平均值。新鲜的PC-3细胞系的RNA值由黄色酒吧和FFPET组织的值示出由彩色点表示。在FFPET前列腺癌样本的DNA（B）甲基化特异性PCR检测。对于由于使用50毫微克/亚硫酸氢盐转化的DNA的反应预计进行10个样品获得循环阈值。所有样品FFPET MSP ALU实验也有类似的周期的阈值（P&gt; 0.67）。 GSTP1 MSP检测表明高甲基化（下周期阈值）在前列腺癌的水平比在前列腺。 （C）的从另外的组织类型的DNA和RNA的质量评价。 HPRT1基因表达和铝基因甲基化测定法从提取的核酸（分别为RNA和DNA）进行，也不发作肝（尸检）和脑（尸检）和乳腺癌（所有FFPET）。从前列腺结果示作比较。注意:从每个组织类型中观察到了相似的结果，除了从一个尸检样品不合格mRNA扩增。每个点或条形代表一个样本，及误差棒表示SEM。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54299/54299fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="12">组织核心</td></tr><tr><td rowspan="2"></td><td rowspan="2">组织输入（MM3） </td><td rowspan="2">提取的核酸</td><td colspan="2">验证</td><td colspan="3">质量检查:DNA </td><td colspan="4">质量检查:RNA </td></tr><tr><td> （样品＃） </td><td>样品的年龄（岁） </td><td>总产出（NG） </td><td>片段大小（BP） </td><html><td> PCR </td><td>总产出（NG） </td><td>片段大小（BP） </td><td> PCR </td><td> NanoString </td></tr><tr><td> Pikor 等。 </td><td> 43 - 129 </td><td>脱氧核糖核酸</td><td>没有数据</td><td>没有数据</td><td>没有数据</td><td>没有数据</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Montaser-Kouhsari 等。 </td><td> 18 - 29.5 </td><td> RNA </td><td> 763 </td><td> 0-25 </td><td></td><td></td><td></td><td> 843 </td><td>没有数据</td><td></td><td></td></tr><tr><td>这张纸</td><td> 1.71 </td><td> DNA和RNA </td><td> &gt; 350 </td><td> 3-12 </td><td> 820 </td><td> 100 - 500 </td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td> 2270 </td><td> 100-500 </td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td>/ftp_upload/54299/check_mark.jpg"/&gt; </td></tr><tr><td colspan="12">组织切片</td></tr><tr><td rowspan="2"></td><td rowspan="2">组织输入</td><td rowspan="2">提取的核酸</td><td colspan="2">验证</td><td colspan="3">质量检查:DNA </td><td colspan="4">质量检查:RNA </td></tr><tr><td> （样品＃） </td><td>样品的年龄（岁） </td><td>总产出（NG） </td><td>片段大小（BP） </td><td> PCR </td><td>总产出（NG） </td><td>片段大小（BP） </td><td> PCR </td><td> NanoString </td></tr><tr><td>海卡尔等。 </td><td> 5×5微米的</td><td>脱氧核糖核酸</td><td> 12 </td><td> 7-22 </td><td> 88-300 </td><td> 103-351 </td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td></td><td></td><td></td><td></td></tr><tr><td> Chung 等。 </td><td> 1×20微米</td><td> RNA </td><td> 9 </td><td> &gt; 5 </td><td></td><td></td><td></td><td> 16,000- 23000 </td><td> 100-200 </td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td></td></tr><tr><td>蒂卡等。 </td><td> 2×4微米</td><td> RNA </td><td> 18 </td><td>没有数据</td><td></td><td></td><td></td><td>未知（621纳克/微升） </td><td> 80-202 + </td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td></td></tr><tr><td> Ghatak 等。 </td><td> 5×5微米的</td><td> DNA和RNA </td><td>五</td><td> 1 </td><td> 14 256 </td><td> &lt;1030 </td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td> 16 000 </td><td> 109-400 + </td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td></td></tr><tr><td>亨尼格等。 </td><td> 1×10微米</td><td> DNA和RNA </td><td> 210 </td><td> 1-25 </td><td>没有数据</td><td>没有数据</td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td>没有数据</td><td>没有数据</td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td></td></tr><tr><td colspan="12">激光捕获显微切割</td></tr><tr><td rowspan="2"></td><td rowspan="2">组织输入（平方毫米） </td><td rowspan="2">提取的核酸</td><td colspan="2">验证</td><td colspan="3">质量检查:DNA </td><td colspan="4">质量检查:RNA </td></tr><tr><td> （样品＃） </td><td>样品的年龄（岁） </td><td>总产出（NG） </td><td>片段大小（BP） </td><td> PCR </td><td>总产出（NG） </td><td>片段大小（BP） </td><td> PCR </td><td> NanoString </td></tR&gt; <tr><td>雪等。 </td><td> 1-2 </td><td>脱氧核糖核酸</td><td> 110 </td><td> 0-2 </td><td> 430 </td><td>没有数据</td><td><img alt="复选标记" src="/files/ftp_upload/54299/check_mark.jpg" /></td><td></td><td></td><td></td><td></td></tr></tbody></table> 表 1: 为组织核心，型材及激光捕获microdissections公布的DNA和RNA提取协议的比较还包括使用PCR和Nanostring测定这些方法几种分子端点评估。

Watch this Scientific Journal Video about Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction at JoVE.com

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

这个修改​​后的提取方法提高了从组织病理学组织块的兴趣更精确的目标区域的RNA和DNA的产量。

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

preparation-formalin-fixed-paraffin-embedded-tissue-cores-for-both

Nanyang Technological University

Research

JoVE Journal

Biology

50.7K 次观看.  Queen's University. 这个修改​​后的提取方法提高了从组织病理学组织块的兴趣更精确的目标区域的RNA和DNA的产量。 

视频: Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

使用Bio - Rad公司的总RNA试剂盒从Neuroprecursor细胞的RNA提取

科研

生物学

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

提取组织抗原的功能分析

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

修正了福尔马林蛋白质组学样品的制备和石蜡包埋组织

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

定量PCR是用于检测巨细胞病毒DNA在福尔马林固定，石蜡包埋的活检组织一个灵敏快速的方法

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...