Name: an improved method of rna isolation from loblolly pine (p. taeda l.) and other conifer species
Uploaded: 2010-02-22T17:00:00
Description: Watch this Scientific Journal Video about An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species at JoVE.com

我们要感谢以下人士没有他们的帮助不会有可能收集的松树组织：乔奈恩博士，马特布里曼，迈克尔波尔多，Ujwal Bagal，靳慧哲，阿曼达Bouffier。

第1部分：树丰收 的树被选中，砍伐后，重要的是那样认真和尽快工作 。 随着每一个不同的组织类型的收获，他们应该立即放置到自己的液氮容器，以避免任何样品材料的交叉污染。 这种RNA隔离协议的可扩展性。 <ol><li>切成约0.5-0.75米长的螺栓和电锯得分在两个或三个地方的树皮螺栓纵轴。 </li><li>使用木凿，工作取得区域的边缘，直到树皮开始从木材?...

<ol>
	<li>Chang, S., Puryear, J., Cairney, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+extracting+RNA+from+pine+trees.">A simple and efficient method for extracting RNA from pine trees.</a> Plant Molecular Biology Reporter. 11 (2), 113-116 (1993).</li><li>Lorenz, W. W., Yu, Y. S., Sim&#369;es, M., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+the+Loblolly+Pine+PtGen2+cDNA+Microarray.">Processing the Loblolly Pine PtGen2 cDNA Microarray.</a> Journal of Visualized Experiments. 25, (2009).</li></ol>

从针叶树种获得高质量的RNA可以是一个艰巨的任务，鉴于木本植物组织中发现的酚类和多糖化合物的高水平。开始与张等人开发的协议。 （1），我们已经发现，更严格的提取和清理步骤导致各种木本和非木本组织从多种针叶树种采样的质量非常高的总RNA的一致隔离。该视频已证明不仅是我们修改后的RNA分离协议，而且在实地收集和火炬松用于RNA分离前处理技术而采取的步骤。这些收获和准备步?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>RNA Isolation Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2% CTAB (hexadecyltrimethylammonium bromide), 2% PVP (polyvinyl pyrrolidinone; Mw 30-40,000), 100mM Tris-HCl (pH8.0), 25mM EDTA, 2.0M NaCl, 0.5g/L Spermidine</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>C298-4</td>
<td>&nbsp;</td>
<tr>
<td>Phenol, Ultrapure</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15509-037</td>
<td>&nbsp;</td>
<tr>
<td>10M LiCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
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<tr>
<td>SSTE Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1M NaCl, 0.5% SDS, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0)</td>
</tr>
<tr>
<td>Sodium acetate (NaOAc) 3M pH4.8</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>Phenol-chloroform (pH8.0)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>120mL phenol, 160mL chloroform titrated with multiple changes of 0.5M Tris-Cl pH 8.0</td>
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<tr>
<td>Oak Ridge High Speed Teflon Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-16B</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 50mL High Speed Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-10K</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon,sterile, capped 50mL conical disposable tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>#21008-939</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Heavy, 2mL</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#2302830</td>
<td>&nbsp;</td>
<tr>
<td>Ambion RNase-free Microfuge Tubes, 2mL</td>
<td>&nbsp;</td>
<td>Ambion, Inc.</td>
<td>#AM12425</td>
<td>&nbsp;</td>		</tbody>
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an improved method of rna isolation from loblolly pine (p. taeda l.) and other conifer species

针叶树种，特别是那些属于松科家族，如火炬松，从组织中分离（<em&gt;火炬松</em&gt; L），含有高浓度的酚类化合物和多糖干扰RNA纯化。从这些品种的高品质的RNA分离需要严格的组织程序，收集多个有机提取步骤以孤立的芯片和其他基因组分析的质量足够的RNA组成的RNA分离协议的领域和就业。从现场收集的火炬松样本可以是具有挑战性的，但标准组织和RNA分离程序的若干修改，大大提高结果的高品质的RNA隔离。如果样品不正确收集和从外地运输，特别是在大型的收成，一般RNA降解程度增加。总RNA产量显着增加，粉碎样品在液氮冷冻轧机前RNA分离，尤其是当从木本植物组织样本。这主要是由于氧化剂的存在，如酚类化合物，多糖，都是在目前大多数针叶树种木本植物组织中提取的高层次，。如果不除去，这些污染物可以开展对领导的问题，如RNA降解，导致产量低和质量差的RNA样品。结转酚类化合物，以及多糖，还可以减少甚至完全消除cDNA合成常用的逆转录酶或其他聚合酶的活性。特别是，RNA的注定用于cDNA文库的产生双链cDNA合成，单链PCR或定量PCR的基因合成，或合成芯片的目标材料的模​​板必须是最高质量，如果研究人员期望以获得最佳的结果。常用于许多其他植物物种的RNA分离技术往往不足以从针叶样品中删除这些污染物的能力，因此不产生总RNA样品，适合于下游操作。在这个视频中，我们展示了针叶树组织进行实地收集的方法，一个四十二岁树砍伐，收获的韧皮部，次生木质部，反应木材木质部开始。我们还证明，一直随后酶操作带来了高品质的RNA的RNA分离协议。

Watch this Scientific Journal Video about An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species at JoVE.com

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

许多植物组织中，包括从火炬松的韧皮部及木质部（<em&gt;火炬松</em&gt; L），含有酚和干扰RNA纯化的多糖高的水平。此演示文稿讨论的收获种植现场组织和隔离的芯片和其他基因组分析的RNA质量足够技术。

的RNA分离方法的改进火炬松（ P。火炬松 L）和其他针叶树种

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

Research

JoVE Journal

Biology

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Processing the Loblolly Pine PtGen2 cDNA Microarray

处理火炬松PtGen2差异表达基因的cDNA微阵列

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

科研

生物学

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

分析甲基化和其他 S腺苷 L</sub捕捉复合质谱（CCMS）>同型半胱氨酸结合蛋白

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Isolation and Purification of Kinesin from Drosophila Embryos

Isolation and Purification of Kinesin from Drosophila Embryos

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

吸血鬼从细胞外液分离<em&gt;秀丽隐杆线虫</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

在对飞行性能的准确，快速测定方法的改进<em&gt;果蝇</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

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 ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

混合式切割：用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

气孔带皮: 一种改进的防护细胞样品制备方法

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...