Name: a novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas
Uploaded: 2014-06-05T14:00:00
Description: Watch this Scientific Journal Video about A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas at JoVE.c

作者要感谢以下美国国立卫生研究院奖项/ subawards对本研究的支持：R01HL106790，R01HL106790-S1，R01HL106786，R01AI089323，R21AI091457，R21RR026006，P20RR020159，C06RR017563，8T32OD011124-08，和P51OD011104。

1，校准Mesodissection仪器与2ID成像软件该2ID成像软件将被称为无论是软件或程序为本文的其余部分。这一步是需要正确地跟踪图像以及对齐多个幻灯片框架。 <ol><li>打开仪器和计算机。 </li><li>打开软件，选择“校准仪器”。 </li><li>通过使用操纵杆校准阶段行程。移动舞台左上角的位置。按“设置”下的程序步骤1。 </li><li>现阶段移动到右下角的位置。按“设置”下的程序步?...

<ol>
	<li>Fend, F., Raffeld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+in+pathology.">Laser capture microdissection in pathology.</a> J Clin Pathol. 53 (9), 666-672 (2000).</li><li>Esposito, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementary+techniques:+laser+capture+microdissection--increasing+specificity+of+gene+expression+profiling+of+cancer+specimens.">Complementary techniques: laser capture microdissection--increasing specificity of gene expression profiling of cancer specimens.</a> Adv Exp Med Biol. 593, 54-65 (2007).</li><li>Adey, N., Bosh, D., Emery, D., Birch, L., Parry, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodissection+of+Paraffin+Embedded+Slide+Mounted+Tissue+Sections.">Mesodissection of Paraffin Embedded Slide Mounted Tissue Sections.</a> Journal of Molecular Diagnostics. 14 (6), (2012).</li><li>Fleischmann, R. D., Alland, D., Eisen, J. A., Carpenter, L., White, O., Peterson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-genome+comparison+of+Mycobacterium+tuberculosis+clinical+and+laboratory+strains.">Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains.</a> J Bacteriol. 184 (19), 5479-5490 (2002).</li><li>Russell, D. G., Barry 3rd, C. E., Flynn, J. 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J., Doyle, L. A., Blanchard, J. L., Lackner, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Granuloma+correlates+of+protection+against+tuberculosis+and+mechanisms+of+immune+modulation+by+Mycobacterium+tuberculosis.">Granuloma correlates of protection against tuberculosis and mechanisms of immune modulation by Mycobacterium tuberculosis.</a> J Infect Dis. 207 (7), 1115-1127 (2013).</li><li>Kaushal, D., Schroeder, B. G., Tyagi, S., Yoshimatsu, T., Scott, C., Ko, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+immunopathology+and+mortality+despite+tissue+persistence+in+a+Mycobacterium+tuberculosis+mutant+lacking+alternative+sigma+factor,+SigH.">Reduced immunopathology and mortality despite tissue persistence in a Mycobacterium tuberculosis mutant lacking alternative sigma factor, SigH.</a> Proc Natl Acad Sci U S A. (12), 8330-8335 (2002).</li><li>Mehra, S., Kaushal, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+reveals+extended+roles+of+the+Mycobacterium+tuberculosis+stress+response+factor+sigmaH.">Functional genomics reveals extended roles of the Mycobacterium tuberculosis stress response factor sigmaH.</a> J Bacteriol. 191 (12), 3965-3980 (2009).</li><li>Rohde, K. 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I., Gomez, M., Tan, D., Pardini, M., Manganelli, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mycobacterium+tuberculosis+sigma+factor+sigmaB+is+required+for+full+response+to+cell+envelope+stress+and+hypoxia+in+vitro,+but+it+is+dispensable+for+in+vivo+growth.">The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth.</a> J Bacteriol. 191 (18), 5628-5633 (2009).</li><li>Rustad, T. R., Harrell, M. I., Liao, R., Sherman, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enduring+hypoxic+response+of+Mycobacterium+tuberculosis.">The enduring hypoxic response of Mycobacterium tuberculosis.</a> PLoS One. 3 (1), (2008).</li></ol>

Mesodissection是可以从所引起的病原体繁多病理病变的幻灯片被应用于RNA提取的技术。它是用户跟踪图像和正确对齐的图像是必要的。要做到这一点，该仪器必须跟随在成像软件的方向进行校正。解剖时，如果被解剖的兴趣幻灯片和面积不对齐，用户必须重新校准仪器。在剥离过程中的另一个重要步骤是加载自耗磨位的方式之前，夹层（步骤5.1），使得它是不含气泡。我们已经发现，气泡的存在降?...

The corresponding author was changed for the article, <a href="http://www.jove.com/video/51693">A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas</a>. The corresponding author was changed from:
Teresa A. Hudock
to:
Deepak Kaushal

The corresponding author was changed for the article, <a href="http://www.jove.com/video/51693">A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas</a>.

Erratum: A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

样品在传统上被手动从任一整个组织或使用针和解剖刀载玻片进行显微解剖。这就需要感兴趣的组织切片与周围组织部分2之间的明确分工。随着分子分析技术目前的进展，出现了越来越需要在细胞水平来评估组织。由于手工显微切割的局限性，已建立的技术包括液晶显示模块，以允许更大的隔离精度。这种技术允许研究者隔离从各种细胞和滑动类型的特定细胞群，然后其可用于下游分析，如基因表达分析。虽然非常有效的下游检测，液晶显示模块也不是没有限制的。首先，LCM是一个昂贵和费时的过程。此外，由于RNA的性质不稳定，它往往是具有挑战性的从LCM样品2获得高质量的RNA。由于diLCM的sadvantages，仍然需要在显微切割技术的新进展，使之更容易在一个合理的时间和敏感的方式更大一些研究人员。 在显微切割技术现在可一个这样的进步是被称为mesodissection的技术。在这种技术中一台机器是用来磨感兴趣的注解组织切片，并吸取它变成一种消耗品磨位3。下一步，该样品被吸入到一个集合管和用于下游应用。这个系统的优点是，它是显著成本较低和时间敏感。在我们的经验中，系统允许在十分钟内肺肉芽肿组织部分的剥离。在另一方面，传统的LCM将可能需要数小时才能完成该过程。该系统允许操作者装入一个参考幻灯片作为比较以及工具注解来使用。另外一个报告生成概述日E区被解剖的幻灯片。有两个主要的缺点mesodissection。虽然在提取多个单元格有效，它是具有挑战性的分离单个细胞。此外，所产生的图像的精度不明确的，因为如果使用其他成像显微镜。 结核病（TB）是人类的一个全球主要的传染病杀手，从感染的结果与结核杆菌 。在大多数暴露于结核分枝杆菌的气溶胶个体，这种感染潜伏的限制。在每年至少 ​​有10万人，它导致活动性肺结核病4。在潜伏感染， 结核杆菌包含称为肉芽肿病理性肺部病灶内。因此，它已被认为结核杆菌感染的结果决定于肉芽肿5的水平。 在这里，我们展示mesodissection如何可以用来microdissect引起结核杆菌肉芽肿。所使用的幻灯片是从被感染的恒河猴FFPE肺组织。此演示的目的，我们将剖析肉芽肿的全部。我们还证明，RNA可从回收的组织被提取。这种技术可以应用于组织样品从各种其他标本，然后用于各种下游检测的。

<table border="0" cellpadding="0" cellspacing="0" width="675">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:260px;">MeSectr</td>
			<td style="width:113px;">AvanSci Bio</td>
			<td style="width:108px;"></td>
			<td style="width:195px;">Mesodissector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">400 nm xScisors</td>
			<td>AvanSci Bio</td>
			<td></td>
			<td>Other sizes available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">THOR</td>
			<td>AvanSci Bio</td>
			<td></td>
			<td>Programmable Heater-Shaker</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NanoDrop2000</td>
			<td>ThermoScientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy FFPE extraction kit&#160;</td>
			<td>Qiagen</td>
			<td align="right" style="width:108px;">73504</td>
			<td style="width:195px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ovation RNA-Seq FFPE System&#160;</td>
			<td>Nugen</td>
			<td align="right">7150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit&#160;</td>
			<td>Qiagen</td>
			<td align="right" style="width:108px;">28104</td>
			<td style="width:195px;"></td>
		</tr>
	</tbody>
</table>

a novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas

显微切割已被用于组织的检查在DNA，RNA和蛋白水平超过十年。激光捕获显微术（LCM）是当今使用的最常见的显微切割技术。在该技术中，激光被用来灶性熔融的热塑性膜，其覆盖一个脱水的组织切片1。组织切片复合，然后抬起并从该膜分离。尽管这种技术可以成功地用于组织检查，这是耗时且昂贵的。此外，在成功完成使用这种技术的程序需要使用的激光的，从而限制了它的使用。所谓mesodissection一个新的更加实惠和实用显微切割方法是一种可行的解决方案，以LCM的陷阱。该技术采用了MESO-1/MeSectr系统轧机从幻灯片上的组织试样所需的组织，而同时分配和吸液至所需的组织样品恢复到耗材厂位。在解剖过程开始时，用户对齐福尔马林固定，石蜡包埋（FFPE）用苏木精伊红染色（H＆E）的参考滑盖滑动。此后，操作者诠释所需的解剖区域，并且前进到解剖相应的段。该程序生成的解剖的存档影像。 mesodissection的主要优点是解剖的滑动所需要的时间短，取平均10分钟从设置采样在该实验中产生。此外，该系统是显著更加成本有效的和用户友好。轻微缺点是，它并不像精确的激光捕获显微术。在这篇文章中我们将演示如何mesodissection可以使用从引起结核分枝杆菌（结核杆菌）FFPE肉芽肿幻灯片中提取的RNA。

上述协议显示了如何使用新的mesodissection技术从FFPE组织切片中提取的RNA。该协议的有效性是通过从NHP肺肉芽肿在各类感染阶段感染了结核杆菌的FFPE幻灯片所示。 图1-3是仪器。 图4的图像描绘了如何在解剖过程中发生，并通过该软件。 表1中生成的结果图像示出的RNA提取与来自NHP肉芽肿在每个感染状态的结果。 RNA扩增和纯化，以允许16秒（ 表2-4）<...

Watch this Scientific Journal Video about A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas at JoVE.c

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

显微切割已被广泛地用于DNA，RNA，和在组织内蛋白质的检查。激光捕获显微术（LCM）是最常用的方法，而是一种新的研磨技术，mesodissection，是最近可用。我们证明RNA提取从mesodissected福尔马林固定，石蜡的结核分枝杆菌肉芽肿包埋组织幻灯片。

一种新型显微切割方法恢复<em&gt;结核分枝杆菌</em&gt;从福尔马林固定，石蜡包埋肺肉芽肿特异性转录

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

Research

JoVE Journal

Immunology and Infection

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

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Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

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Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

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Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...

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分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

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Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...