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.
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.
Part 2: Freezer Mill Processing of Samples
Part 3: RNA Isolation, Day 1
Part 4: RNA Isolation, Day 2
Part 5: RNA Precipitation and Ethanol Wash
Part 6: Final Resuspension and Quantification
Representative Results:
RNA isolation yields from different conifer tissues range from 60-120 μg/g frozen tissue with woody samples typically yielding around 70-80g/g frozen tissue. Diagnostic ratios of 260/280 and 260/230 are both typically greater than 2.1, and are good indicators that the RNA sample is free of any significant phenolic or carbohydrate contamination, respectively. Samples analyzed by agarose gel electrophoresis followed by EtBr staining should show three distinct bands for 25S, 18S, and 5S RNA (Figure 1). Determining the stoichiometry of the 28S to 18S on agarose gels is somewhat subjective, and factors such as running conditions for the gel, staining, and sample load can all have an effect. In general, the 28S:18S ratio will appear to be > 1.5. However, some conifer samples have lower ratios. For example, following Agilent 2100 analysis we have seen several instances of conifer samples from different tissue types where the stoichiometry is closer to 1.2:1. Thus, an apparent low 28S:18S stoichiometry by agarose gel electrophoresis does not necessarily indicate a poor-quality sample. Leaf, shoot, and cone samples will often have additional distinct bands present due to chloroplastic ribosomal RNAs. EtBr-stained materials that do not migrate from the sample well or high molecular mass bands (>8-10 kb) usually indicate genomic DNA contamination (Figure 2). Low molecular mass smears in the vicinity of or below the 5S band and reduced ribosomal band staining or an apparent 18S > 28S stoichiometry is a good indicator that RNA degradation has occurred (Figure 3). In the Agilent 2100 electropherogram analysis, the base line should be low and relatively smooth with major peaks corresponding to 18S and 28S ribosomal RNAs having 28S:18S ratios ranging anywhere from 1.2 to 2.0. The Agilent RNA Integrity Number (RIN) value may be a better predictor of RNA quality and should be at least 7 or greater for RNA that is to be used for the preparation of microarray targets. Figure 4 shows a typical Agilent 2100 electropherogram for xylem RNA with a 28S:18S ratio equal to 1.4 and a RIN value of 8.6 while Figure 5 shows a poor xylem RNA prep having a very high baseline and noticeable degradation as revealed by the 28S:18S ratio equal to 0.65 and a RIN value of 3.4.
Figure 1. Agarose gel analysis of high-quality pine RNA. Lane 1, NEB 1 kb standard, Lane 2 shows a typical total RNA isolation from loblolly pine secondary xylem with characteristic ribosomal 28S, 18S, and 5S bands and apparent 28S:18S ratio >1.
Figure 2.Agarose gel analysis of pine RNA sample contaminated with genomic DNA. Lane 1, NEB 1 kb standard, Lane 2, xylem RNA sample showing genomic DNA contamination.
Figure 3.Agarose gel analysis of pine sample showing RNA degradation and contamination with genomic DNA. Lane 1, NEB 1 kb standard, Lane2, xylem RNA sample showing genomic DNA contamination and severe RNA degradation.
Figure 4.Agilent 2100 electropherogram of xylem total RNA isolated using the described method on loblolly pine xylem. 28S:18S ratio equals 1.4, RIN value equals 8.6
Figure 5.Agilent 2100 electropherogram of apical tip total RNA showing severe degradation and genomic contamination. 28S:18S ratio equals 0.65, RIN value equals 3.4
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 steps taken in field collection and processing techniques used for loblolly pine prior to RNA isolation. These harvesting and preparation steps are equally important to minimize RNA degradation in field-collected samples, as well as to increase both RNA quality and total yield. Most significantly, we have found that RNA prepared using this method has led to increased cDNA synthesis yields compared to other methods used to prepare microarray targets for hybridization against the PtGen2 loblolly pine microarray (2).
We would like to thank the following persons without whose help the collection of pine tissues would not have been possible: Dr. Joe Nairn, Matt Bryman, Michael Bordeaux, Ujwal Bagal, Huizhe Jin, and Amanda Bouffier.
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 |