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

डा. जो Nairn, मैट Bryman, माइकल बोर्डो, Ujwal Bagal, Huizhe जिन, और अमांडा Bouffier: हम बिना निम्नलिखित व्यक्तियों जिनकी मदद पाइन ऊतकों के संग्रह संभव नहीं होता को धन्यवाद देना चाहूंगा.

भाग 1: ट्री हार्वेस्ट बाद पेड़ चयनित और felled है, यह महत्वपूर्ण है सावधानी के रूप में और जितना जल्दी संभव हो काम. प्रत्येक अलग ऊतक प्रकार के रूप में काटा जाता है, वे अपने स्वयं के तरल नाइट्रोजन वाह...

<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>

शंकुवृक्ष प्रजातियों से प्राप्त उच्च गुणवत्ता वाले आरएनए phenolic और polysaccharide वुडी ऊतकों में पाया यौगिकों के उच्च स्तर को देखते हुए एक मुश्किल काम हो सकता है. चांग एट अल द्वारा विकसित प्रोटोकॉल के साथ शुरू. (1), ह...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<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>
</tr>
<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>
</tr>
<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>
		</table>
		</div>

an improved method of rna isolation from loblolly pine (p. taeda l.) and other conifer species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

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

फ्लोएम और विनोदी पाइन से जाइलेम सहित कई संयंत्र के ऊतकों (<em&gt; Pinus taeda</em&gt;) एल, phenolics और polysaccharides कि शाही सेना शुद्धि के साथ हस्तक्षेप के उच्च स्तर होते हैं. इस प्रस्तुति क्षेत्र में विकसित ऊतकों और प्रोटीन और अन्य जीनोमिक विश्लेषण के लिए पर्याप्त गुणवत्ता की शाही सेना के अलगाव की फसल के लिए तकनीक चर्चा.

विनोदी पाइन से एक शाही सेना के अलगाव की बेहतर विधि ( पी. taeda एल) और अन्य शंकुवृक्ष प्रजाति

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

प्रसंस्करण विनोदी पाइन PtGen2 सीडीएनए माइक्रोएरे

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

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

भ्रूणीय Zebrafish और सीडीएनए संश्लेषण से जीन एक्सप्रेशन विश्लेषण के लिए शाही सेना अलगाव

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

Methyltransferases की रूपरेखा और अन्य एस Adenosyl एल</subकैद यौगिक मास स्पेक्ट्रोमेट्री (CCMS)> homocysteine ​​बाध्यकारी प्रोटीन

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

एक अवेध्य बालों नमूनाकरण मायावी छोटे स्तनधारी से उच्च गुणवत्ता डीएनए प्राप्त तकनीक

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

अलगाव और kinesin के शोधन से ड्रोसोफिला भ्रूण

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

पैशाचिक कोशिकेतर द्रव से अलगाव<em&gt; Caenorhabditis एलिगेंस</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

उड़ान प्रदर्शन का सही और तेजी से मापन के लिए एक बेहतर तरीका में<em&gt; ड्रोसोफिला</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

माउस अग्न्याशय से शाही सेना अलगाव: एक ribonuclease युक्त ऊतक

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

हाइब्रिड-कट: अड़ियल संयंत्र के ऊतकों के नमूनों के लिए एक बेहतर सेक्शनिंग विधि

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

Stomata टेप-पील: गार्ड सेल नमूना तैयारी के लिए एक बेहतर विधि

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 ...