Rapid High Quality RNA Preparation from Pine Seedlings

Conventional RNA extraction methods have been shown to produce poor quality RNA when applied to pine and other gymnosperms. We present a protocol for extracting highly pure RNA from pine. Modifications to conventional procedures include: 1) the use of seedlings, 2) the use of phenol and PVP to rapidly remove DNA, proteins and pigments, and 3) the use of salt precipitation to remove other contaminating compounds. The procedure can be completed in less than eight hours. Yield and purity were monitored by denaturing gel electrophoresis and by UV absorbance (A₂₆₀ /A₂₈₀ and A₂₆₀/A₂₃₀). These ratios were over 2, indicating an absence of contaminating metabolites. Additionally, a new absorbance ratio (A₂₆₀/A₂₁₀) was introduced to monitor the RNA purity in each step (it indicates the ratio of covalent links in the solution belonging to RNA). The yield was around 300 µg total RNA per gram of tissue of Pinus sylvestris and over 400 µg of total RNA per gram of P. pinaster tissue, which is a high recovery (more than 63%) for gymnosperms. The RNA was of sufficient quality for use in a RT-PCR reaction that amplified 1 kb of the pine GS gene. This protocol has been applied with success to other woody plants like Populus species.Abbreviations: DEPC, diethyl pyrocarbonate; AMV, avian myeloblastosis virus; FW, fresh weight.     

Characterization of wheat root ribosomes isolated by Mg2+ precipitation

The Mg²⁺ precipitation method has been adapted for isolation of ribosomes from roots of wheat. The ribosomes prepared by this procedure show A₂₆₀/A₂₈₀ = 1.6 and A₂₆₀/A₂₃₅ = 1.3 and contain 44d% RNA and 56% ribosomal proteins. There are no detectable differences in the ribosomal protein complement and accessibility of the ribosomal proteins to phosphorylation between ribosomes isolated by this procedure and those prepared by classical ultracentrifugation methods. The ribosomes are active in a poly-U directed cell-free system for protein synthesis.     

RNA isolation from loquat and other recalcitrant woody plants with high quality and yield

RNA isolation is difficult in plants that contain large amounts of polysaccharides and polyphenol compounds. To date, no commercial kit has been developed for the isolation of high-quality RNA from tissues with these characteristics, especially for fruit. The common protocols for RNA isolation are tedious and usually result in poor yields when applied to recalcitrant plant tissues. Here an efficient RNA isolation protocol based on cetyltrimethylammonium bromide (CTAB) and two successive precipitations with 10 M lithium chloride (LiCl) was developed specifically for loquat fruits, but it was proved to work efficiently in other tissues of loquat and woody plants. The RNA isolated by this improved protocol was not only of high purity and integrity (A₂₆₀/A₂₈₀ ratios ranged from 1.90 to 2.04 and A₂₆₀/A₂₃₀ ratios were > 2.0) but also of high yield (up to 720 μg on average [coefficient of variation = 21%] total RNA per gram fresh tissue). The protocol was tested on loquat fruit (different stages of development, postharvest, ripening, and bruising), leaf, root, flower, stem, and bud; quince fruit and root; grapevine cells in liquid culture; and rose petals. The RNA obtained with this method is amenable to enzymatic treatments and can be efficiently applied for research on gene characterization, expression, and function.     

Co-isolation of high-quality DNA and RNA from coenocytic green algae

A protocol is presented for the simultaneous isolation of DNA and RNA from giant-celled green algae. The overall quality of the DNA was examined by the A²⁶⁰/A²⁸⁰ ratio, agarose gel electrophoresis, and restriction enzyme analysis. Denaturing gel electrophoresis and cDNA cloning were used to investigate the quality of the RNA. These assays indicated that both the DNA and RNA isolated by this procedure are of high quality, suitable for further molecular analyses. Since many of these algae are slow growing and therefore only a few grams may be available, the isolation of DNA and RNA from the same plant material has obvious advantages.Abbreviations: Etbr, ethidium bromide.     

Improved RNA isolation from Laminaria japonica Aresch (Laminariaceae, Phaeophyta)

RNA isolation is difficult in some plants and algae because phenolics, polysaccharides, or other compounds can bind or co-precipitate with RNA, and because the success of RNA isolation can be strain-specific and species-specific. To create an improved RNA isolation protocol for Laminaria japonica Aresch (Laminariaceae, Phaeophyta), four methods for extracting RNA were tested. A cetyltrimethylammonium bromide (CTAB)-based RNA extraction protocol was developed that clearly showed 28S and 18S ribosomal RNA bands and produced RNA with high yield (68 μg g⁻¹ fresh weight) and high quality (A _260/280 ratio 1.96 ± 0.05). The isolated RNA was intact, and RT-PCR analysis confirmed that further molecular application is feasible.     

Elevated CO2 affects photosynthetic responses in canopy pine and subcanopy deciduous trees over 10 years: a synthesis from Duke FACE

Leaf responses to elevated atmospheric CO₂ concentration (C_a) are central to models of forest CO₂ exchange with the atmosphere and constrain the magnitude of the future carbon sink. Estimating the magnitude of primary productivity enhancement of forests in elevated C_a requires an understanding of how photosynthesis is regulated by diffusional and biochemical components and up‐scaled to entire canopies. To test the sensitivity of leaf photosynthesis and stomatal conductance to elevated C_a in time and space, we compiled a comprehensive dataset measured over 10 years for a temperate pine forest of Pinus taeda, but also including deciduous species, primarily Liquidambar styraciflua. We combined over one thousand controlled‐response curves of photosynthesis as a function of environmental drivers (light, air C_a and temperature) measured at canopy heights up to 20 m over 11 years (1996–2006) to generate parameterizations for leaf‐scale models for the Duke free‐air CO₂ enrichment (FACE) experiment. The enhancement of leaf net photosynthesis (A_net) in P. taeda by elevated C_a of +200 μmol mol^?1 was 67% for current‐year needles in the upper crown in summer conditions over 10 years. Photosynthetic enhancement of P. taeda at the leaf‐scale increased by two‐fold from the driest to wettest growing seasons. Current‐year pine foliage A_net was sensitive to temporal variation, whereas previous‐year foliage A_net was less responsive and overall showed less enhancement (+30%). Photosynthetic downregulation in overwintering upper canopy pine needles was small at average leaf N (N_area), but statistically significant. In contrast, co‐dominant and subcanopy L. styraciflua trees showed A_net enhancement of 62% and no A_net–N_area adjustments. Various understory deciduous tree species showed an average A_net enhancement of 42%. Differences in photosynthetic responses between overwintering pine needles and subcanopy deciduous leaves suggest that increased C_a has the potential to enhance the mixed‐species composition of planted pine stands and, by extension, naturally regenerating pine‐dominated stands.     

A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.)

Many experiments in plant molecular biology require processing of a large number of RNA samples and in some cases large quantities are required for a single application. In turmeric, a major spice and medicinal plant, a protocol for RNA isolation is not available. The major difficulty encountered while using other popular protocols is the low yield and quality of RNA which hampers the downstream applications like qRT-PCR, cDNA synthesis and micro RNA isolation. Commercial kits though available are costly and were found to be unsuccessful in case of rhizomes and root tissues that are rich in polyphenols, polysaccharides and alkaloids. It was thus felt that a quick, handy and cheap protocol of total RNA isolation from different tissues of turmeric was required for day to day working in our lab. The new protocol utilizes SDS based extraction buffer including β-mercaptoethanol and PVP with sequential acid phenol:chloroform extraction to remove polyphenols and proteins, followed by the purification with sodium acetate to eliminate polysaccharides. The protocol is simple and can be completed in less than 3 h. The RNA yield from rhizome was higher by more than fivefold with both A_260/280 and A_260/230 ratio in the range of 1.8–2.0. The protocol worked well with leaf, rhizome, pseudostem and root tissues with RIN >7.0 and the isolated RNA could be successfully used for cDNA synthesis, RT-PCR, qRT-PCR and small RNA isolation including microRNA.     

A new cell for microspectrophotometry and its use in studying tRNA-Leu-CUG from Escherichia coli

tRNA_CUG^Leu is believed not to contain 4-thiouridine, but its absorption spectrum contains a peak at 338 nm of magnitude about 1% of A₂₆₀. This has been verified by carrying out serial dilutions of dissolved crystalline material, using a microspectrophotometer with a new cell requiring only 1.5 μl for a path length of 3 mm. Crystallizable tRNA^Phe and tRNA_GUA.G^Val show normal 4-thiouridine peaks of magnitude 2% of A₂₆₀, so the anomalous spectrum seems unlikely to be an artefact of the isolation procedure used for all three species.     

Isolation of RNA of high quality and yield from <Emphasis Type="BoldItalic">Ginkgo biloba</Emphasis> leaves

An improved protocol was developed to isolate total RNA in good yield and integrity from Ginkgo biloba leaves containing high levels of flavonoid glycosides, terpene lactones, carbohydrates and polyphenolic secondary metabolites. Polyvinylpolypyrrolidone at 2% and β-mercaptoethanol at 4% were added to the standard CTAB extraction buffer and, after chloroform and phenol extraction, the pellet obtained by ethanol/acetate precipitation was washed and a second phenol/chloroform extraction was introduced to remove co-precipitated polysaccharides. Both A₂₆₀/A₂₃₀ and A₂₆₀/A₂₈₀ absorbancy ratios of isolated RNA were around 2 and the yield was about 0.4 mg g^--1 fresh weight. At least seven distinct rRNA bands were detected by denaturing gel electrophoresis. Sharp hybridization signals were obtained from Northern blots with both nuclear and plastid gene probes. Two gene fragments: nuclear-encoded cab and chloroplast encoded rbcL were successfully amplified by RT-PCR, suggesting the integrity of isolated RNA. The total RNA isolated by this protocol is of sufficient quality for subsequent molecular applications.     

Relationship between net photosynthesis and nitrogen in Scots pine: Seasonal variation in seedlings and shoots

The relationship between light saturated net photosynthesis (A_max) and nitrogen concentration (N) was studied in needles of both Scots pine seedlings, grown at three relative growth rates (2,6 and 8%) controlled by nutrient addition rate, and Scots pine shoots collected from four sites with different fertility. In the seedlings, A_max was measured on 14 different dates starting at the beginning of the second growing season and ending when growth of the new shoot and the secondary needles had finished. In shoots from the natural stands A_max of the previous-year shoots was measured on 6 dates throughout the growing season.Both in seedlings and shoots, the correlation between A_max and N was poor, when data from all sampling dates were taken together. However, A_max was correlated with N in most instances when the age of the needles was considered and the data were examined either at weekly intervals (seedlings) or separately for each sampling date (shoots). The slope of the A_max vs N relationship varied greatly between sampling dates. In the seedlings the correlation between A_max and N was strongest by the time when the new needles were developing. In the shoots the correlation was significant from mid June until mid August, while no correlation was found in the beginning and at the end of the growing season.Our data indicate that in pine needles the photosynthesis-nitrogen relationship is more complex than in broadleaved species. Contrary to the broadleaved species, where the correlation is independent of sampling time, in this conifer the time of the year affects the correlation and there are phases during the growing season when the correlation is poor or nonexistent.Abbreviations A_max light saturated net photosynthesis - kPa kilopascal - N nitrogen concentration in the needles - PFD photon flux density (400–700 nm) - RGR relative growth rate     

Rapid and Effective Method of RNA Isolation from Green Microalga <Emphasis Type="Italic">Ankistrodesmus convolutus</Emphasis>

The rapid and effective method for the isolation of RNA from green microalga Ankistrodesmus convolutus based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride is developed. This procedure avoids the use of toxic chaotropic agents and phenol while high concentration of dithiothreitol is used to inhibit RNase activity and prevent oxidative cross-linking of nucleic acids by phenolics. The extraction procedure was able to produce high quality and intact RNA from A. convolutus. The yield of total RNA was 0.69–0.73 mg/g of fresh weight, with A₂₆₀/A₂₈₀ ratio of 1.79–1.86. The obtained RNA was of sufficient quality and suitable for downstream application such as RT-PCR and cDNA library construction. The procedure may also have wider applicability for total RNA isolation from other green microalgae species.     

Efficient isolation of high quality nucleic acids from different tissues of <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Improved and efficient methods were developed for isolating high quality DNA and RNA from different sources of Iranian Yew (Taxus baccata L.). The methods were based on CTAB extraction buffer added with high levels of polyvinylpyrrolidone (PVP) and β-mercaptoethanol to properly remove polysaccharides and prevent oxidation of phenolics. The pellets obtained by ethanol precipitation were washed only with Chloroform: isoamyl alcohol (24:1). So, we could successfully eliminate the dangerous phenol/chloroform extraction steps from the isolation procedure. Both spectrophotometric (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios) and agarose electrophoresis analysis of isolated nucleic acids (DNA and RNA) indicated good results. DNA with the average yield of 100–300 μg/g leaf and stem tissue and total RNA with an average yield of 20–30 μg/g cell culture and 80–100 μg/g leaf and stem tissue of Iranian yew could be obtained. Successful amplification of pam and pds by PCR and RT-PCR, showed the integrity of isolated DNA and RNA, respectively.     

Effect of open-air fumigation with sulphur dioxide and ozone on phyllosphere and endophytic fungi of conifer needles

The phyllosphere microbial populations inhabiting the needles of three conifer species, Scots pine (Pinus sylvestris L.), Sitka spruce (Picea sitchensis L.) and Norway spruce (Picea abies (L.) Karst.), exposed to SO₂ and O₃, in an open-air fumigation experiment were analysed over a 3 year period using serial dilution after washing, direct plating and a fluorescein diacetate (FDA) enzyme assay. Total fungal populations ranged from 10² to 10⁵ colonyforming units (CPU) g^?1 fresh weight of needles. The dominant fungi isolated from needles varied with tree species and isolation technique; Aureobasidium pullulans (de Bary) Arnaud was most common on Scots pine and Norway spruce and white yeasts on Sitka spruce using the dilution plating method. However, direct plating of needle segments onto culture media indicated that Sclerophoma pythiophila (Corda) Hohnel was dominant on Scots pine and A. pullulans on Sitka and Norway spruce. Green needles of Sitka spruce were found to be endophytically colonized by Rhizosphaera kalkhoffii Bubak, but seldom by Lophodermium piceae (Fuckel) Hohn during extensive sampling in 1990. Statistical analyses revealed significant differences (P<0.05) between plots in the 3 year mean of the total fungal populations or the fungal biomass (FDA assay) on all three tree species. Differences between plots were also observed for a number of dominant component species. Data were also analysed for treatment effects. A significant effect of SO₂ treatment was observed on the total fungal populations on Sitka spruce (P<0.05) which were reduced markedly by the low-SO₂ treatment, while the O₃ treatment caused a significant increase in total fungal numbers on Scots pine (P<0.05). The FDA activity on needles of both Scots pine and Sitka spruce was noticeably higher in the 0₃-only treatment plot, but the overall O₃ effect was not significant. Treatment effects were also detected on the occurrence of component species. The serial dilution method revealed an SO₂ effect (P<0.05) of a reduction in the occurrence of pink yeasts on Sitka spruce and an O₃ effect (P<0.05) of an increase in the occurrence of S. pythiophila on Sitka spruce (P<0.01) but a decrease of Epicoccum nigrum Link and Cladosporium spp. on Scots pine. The direct-plating method revealed an SO₂ effect of an increase in S. pythiophila on Norway spruce (P<0.05). Ozone treatment caused a significant increase in the isolation of a black strain of A. pullulans on Norway spruce (P<0.05). Endophytic colonization of Sitka spruce needles by R. kalkhoffii was found to be increased on two occasions by O₃ exposure.     

A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites

A protocol is described for rapid RNA isolation from various plant species and tissues rich in polyphenolics and polysaccharides. The method is based on the Nucleon PhytoPure^™ system without the use of phenol. The procedure can be completed within 1 h and many samples can be processed at the same time. The yield ranged from 240 μg up to 3 mg per gram of tissue with an average purity measured as A_260/280 of 1.85. The RNA was of sufficient quality for use in RT-PCR reactions. Quantitation of single-stranded cDNA was carried out with the RiboGreen^™ reagent and of PCR products with the PicoGreen^™ reagent.