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

Summary

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

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

Part 1: Tree Harvest

After the tree is selected and felled, it is important to work as carefully and as quickly as possible. As each different tissue type is harvested, they should be placed immediately into their own liquid nitrogen vessels to avoid any cross contamination of sample material. This RNA isolation protocol is scalable.

1. Cut bolts into lengths of approximately 0.5-0.75 m and with a chainsaw score the bark in two or three places following the longitudinal axis of the bolt.
2. Using a wood chisel, work the edge of the scored region until the bark begins to separate from the wood and continu....

Discussion

Obtaining high-quality RNA from conifer species can be a difficult task given the high levels of phenolic and polysaccharide compounds found in woody tissues. Starting with the protocol developed by Chang et al. (1), we have found that more rigorous extraction and clean up steps lead to the consistent isolation of very high-quality total RNA from various woody and non-woody tissues sampled from a wide variety of conifer species. This video has demonstrated not only our modified RNA isolation protocol, but also the step.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 Scientific, Inc.	#05-562-16B
Polypropylene 50mL High Speed Tubes	Thermo Fisher Scientific, Inc.	#05-562-10K
BD Falcon,sterile, capped 50mL conical disposable tubes	VWR international	#21008-939
Phase Lock Gel Heavy, 2mL	Thermo Fisher Scientific, Inc.	#2302830
Ambion RNase-free Microfuge Tubes, 2mL	Ambion	#AM12425