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

Summary

फ्लोएम और विनोदी पाइन से जाइलेम सहित कई संयंत्र के ऊतकों (<em> Pinus taeda</em>) 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.

Abstract

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.

Protocol

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…

Discussion

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…

Materials

Material Name	Type	Company	Catalogue Number	Comment
RNA Isolation Buffer				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
Chloroform		Fisher Scientific	C298-4
Phenol, Ultrapure		Invitrogen	15509-037
10M LiCl				Made with DEPC-treated water
SSTE Buffer				1M NaCl, 0.5% SDS, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0)
Sodium acetate (NaOAc) 3M pH4.8				Made with DEPC-treated water
Phenol-chloroform (pH8.0)				120mL phenol, 160mL chloroform titrated with multiple changes of 0.5M Tris-Cl pH 8.0
Oak Ridge High Speed Teflon Tubes		Thermo-Fisher	#05-562-16B
Polypropylene 50mL High Speed Tubes		Thermo-Fisher	#05-562-10K
BD Falcon,sterile, capped 50mL conical disposable tubes		VWR	#21008-939
Phase Lock Gel Heavy, 2mL		Thermo-Fisher	#2302830
Ambion RNase-free Microfuge Tubes, 2mL		Ambion, Inc.	#AM12425