An improved method for the isolation of total RNA from Avicennia germinans leaves

Isolation of high-quality RNA of Avicennia germinans L. tissue is difficult due to high levels of phenols and other substances that interfere when using conventional procedures for the isolation. These substances not only decrease the yield but also the quality of RNA is almost poor. We present here a simple RNA protocol and fast methodology that effectively removes these contaminating substances without affecting the yield. The protocol developed is based on the SDS/phenol method with modifications including beta-mercaptoethanol to prevent oxidation of phenolic complexes, and phenol/chloroform extraction is introduced to remove proteins, genomic DNA, and secondary metabolites, and co-precipitated polysaccharides. Both A260/A230 and A260/A280 absorbance ratios of isolated RNA were around 2 and the yield was about 0.3 mg g(-1) fresh weight. Good-quality total RNA from leaves of Avicennia germinans could be easily isolated within 2 h by this protocol which avoided the limitation of plant materials and could provide total RNA for all kinds of further molecular studies.     

Isolation of High-Quality RNA from <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>, a Desert Plant Rich in Secondary Metabolites

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.     

Development of an Efficient Protocol of RNA Isolation from Recalcitrant Tree Tissues

Isolation of RNA from recalcitrant tree tissues has been problematic due to large amounts of secondary metabolites and interfering compounds in their cells. We have developed an efficient RNA extraction method, which yielded high-quality RNA preparations from tissues of the lychee tree. The method reported here utilized EDTA, LSS, and CTAB to successfully inhibit RNase activities. It was found that a high ionic strength brought about by 2 M NaCl was necessary. In addition, secondary metabolites and other interfering compounds were effectively removed using sodium borate and PVPP under a deoxidized condition. The quality of purified RNA was tested by both RACE and Northern blotting analysis, ensuring that the RNA could be used for subsequent gene expression analysis. This method has been successfully applied to purify RNA from 15 other plant species. In conclusion, the protocol reported here is expected to have excellent applications for RNA isolation from recalcitrant plant tissues.     

A robust,cost‐effective method for DNA,RNA and protein co‐extraction from soil,other complex microbiomes and pure cultures

The soil microbiome is inherently complex with high biological diversity, and spatial heterogeneity typically occurring on the submillimetre scale. To study the microbial ecology of soils, and other microbiomes, biomolecules, that is, nucleic acids and proteins, must be efficiently and reliably co‐recovered from the same biological samples. Commercial kits are currently available for the co‐extraction of DNA, RNA and proteins but none has been developed for soil samples. We present a new protocol drawing on existing phenol–chloroform‐based methods for nucleic acids co‐extraction but incorporating targeted precipitation of proteins from the phenol phase. The protocol is cost‐effective and robust, and easily implemented using reagents commonly available in laboratories. The method is estimated to be eight times cheaper than using disparate commercial kits for the isolation of DNA and/or RNA, and proteins, from soil. The method is effective, providing good quality biomolecules from a diverse range of soil types, with clay contents varying from 9.5% to 35.1%, which we successfully used for downstream, high‐throughput gene sequencing and metaproteomics. Additionally, we demonstrate that the protocol can also be easily implemented for biomolecule co‐extraction from other complex microbiome samples, including cattle slurry and microbial communities recovered from anaerobic bioreactors, as well as from Gram‐positive and Gram‐negative pure cultures.     

Modified Cetyltrimethylammonium bromide method improves robustness and versatility: The benchmark for plant RNA extraction

A wide range of plant RNA extraction methods are available; however, many of these are limited in their application for a diverse range of plant species. With special emphasis on robustness and versatility, we have improved the cetyltrimethylammonium bromide (CTAB) method and isolated high-quality RNA from 16 different plant species. The major modifications made to the protocol described here were a reduction of sample treatment steps and an increase in β-mercaptoethanol concentration (to 3%) resulting in a robust, rapid and reproducible plant RNA extraction protocol that can be used for a broad range of plant species and tissue types.     

Mitochondrial DNA and RNA isolation from small amounts of potato tissue

We present a fast and simple protocol for purification of mitochondrial DNA and RNA from small amounts of potato tissue including tubers, leaves, flowers, and flower buds. This method uses a high ionic strength medium to isolate mitochondria and extract mitochondrial DNA and RNA from a single preparation and is easily adaptable to other plant species. The mitochondrial DNA was not contaminated by plastid DNA, was fully restrictable and was successfully used for PCR, cloning and Southern analyses. Similarly, the isolated mitochondrial RNA was not contaminated (flower buds) or only slightly contaminated (leaves) by plastid RNA. RNA prepared according to our method was acceptable for northern and RT-PCR analyses.     

Rapid isolation of high-quality total RNA from taxus and ginkgo

An easy and efficient protocol was developed for isolating good-quality total RNA from various tissues including fruits, leaves, stems, and roots of ancient gymnosperm species, taxus and ginkgo. The protocol was developed based on the CTAB method with modifications, including higher-strength CTAB to help the lysis of plant cells, more PVP, and beta-mercaptoethanol to prevent oxidation of phenolic complexes, and higher-centrifugation force to get rid of most cell debris and to ensure RNA quality. In RNA isolation, chloroform/isoamyl alcohol was used to remove proteins, genomic DNA, and secondary metabolites and lithium chloride was subsequently adopted to concentrate total RNA away from most of the cytoplasmic components. Good-quality total RNA from various tissues of native taxus and ginkgo could be easily isolated within 24 hr by this protocol which avoided the limitation of plant materials and the usage of dangerous chemicals, such as phenol, and could provide total RNA for all kinds of further molecular studies.     

Elimination of oxalate contamination in RNA isolation from<Emphasis Type="Italic">Rumex obtusifolius</Emphasis>

Many plant RNA isolation techniques aim to prevent contamination by means of secondary phenolics, carbohydrates, RNase, and other chemicals. However, when applied in our laboratory to the isolation of RNA fromRumex obtusifolius, these protocols failed to produce good quality RNA. A major problem was contamination of the RNA samples with the secondary metabolite oxalate. The relative quantities of guanidine isothiocyanate extraction buffer to plant tissue used in the protocol had significant effects on oxalate contamination. An increase in extraction buffer, from 1.5 mL in the original method to 15 mL per 200–300 mg of tissue in our protocol, removed the oxalate from the RNA. This RNA was of a good quality and was suitable for molecular biology applications.     

Optimization of DNA extraction from seeds and leaf tissues of Chrysanthemum (Chrysanthemum indicum) for polymerase chain reaction

Chrysanthemums constitute approximately 30 species of perennial flowering plants, belonging to the family Asteraceae, native to Asia and Northeastern Europe. Chrysanthemum is a natural cosmetic additive extracted from Chinese herb by modern biochemical technology. It has the properties of anti-bacterial, anti-viral, reducing (detoxification) and anti-inflammation. It possesses antioxidant characteristics, which could assist in minimizing free-radical induced damage. Therefore, it is widely used in skin and hair care products. Chemical composition of this herbal remedy includes kikkanols, sesquiterpenes, flavonoids, various essential oils containing camphor, cineole, sabinol, borneole and other elements that interfere with DNA, causing erroneous or no PCR products. In the present study, testing and modification of various standard protocols for isolation of high-quality DNA from leaf tissues and seeds of C. indicum was done. It was observed that the DNA obtained from seeds and leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. Also, DNA could be extracted from leaf tissues without using liquid nitrogen. Quality of DNA extracted from seeds was much better as compared to that extracted from leaf tissues. The extracted DNA was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extraction of high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils.     

Protein Extraction From Leaves of Aloe vera L., A Succulent and Recalcitrant Plant, for Proteomic Analysis

An improved method for extracting proteins from leaf tissues of Aloe vera L., a recalcitrant plant species, for proteomic analysis is presented. In this protocol, the following critical components are included. A washing step is added prior to homogenization of the tissue to eliminate contaminants, and a concentrated 2× extraction buffer (pH 7.5) is used to increase protein yield. Compared to classical trichloroacetic acid–acetone and phenol extraction methods, this novel protocol has yielded two-dimensional electrophoresis gels with minimal (if any) streaking and provided high-quality protein samples. This protocol is expected to be applicable to other recalcitrant plant tissues.     

A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses

Prunus persica has been proposed as a genomic model for deciduous trees and the Rosaceae family. Optimized protocols for RNA isolation are necessary to further advance studies in this model species such that functional genomics analyses may be performed. Here we present an optimized protocol to rapidly and efficiently purify high quality total RNA from peach fruits (Prunus persica). Isolating high-quality RNA from fruit tissue is often difficult due to large quantities of polysaccharides and polyphenolic compounds that accumulate in this tissue and co-purify with the RNA. Here we demonstrate that a modified version of the method used to isolate RNA from pine trees and the woody plant Cinnamomun tenuipilum is ideal for isolating high quality RNA from the fruits of Prunus persica. This RNA may be used for many functional genomic based experiments such as RT-PCR and the construction of large-insert cDNA libraries.     

Rapid and Efficient Isolation of High-Quality Small RNAs from Recalcitrant Plant Species Rich in Polyphenols and Polysaccharides

Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are important regulators of plant development and gene expression. The acquisition of high-quality small RNAs is the first step in the study of its expression and function analysis, yet the extraction method of small RNAs in recalcitrant plant tissues with various secondary metabolites is not well established, especially for tropical and subtropical plant species rich in polysaccharides and polyphenols. Here, we developed a simple and efficient method for high quality small RNAs extraction from recalcitrant plant species. Prior to RNA isolation, a precursory step with a CTAB-PVPP buffer system could efficiently remove compounds and secondary metabolites interfering with RNAs from homogenized lysates. Then, total RNAs were extracted by Trizol reagents followed by a differential precipitation of high-molecular-weight (HMW) RNAs using polyethylene glycol (PEG) 8000. Finally, small RNAs could be easily recovered from supernatant by ethanol precipitation without extra elimination steps. The isolated small RNAs from papaya showed high quality through a clear background on gel and a distinct northern blotting signal with miR159a probe, compared with other published protocols. Additionally, the small RNAs extracted from papaya were successfully used for validation of both predicted miRNAs and the putative conserved tasiARFs. Furthermore, the extraction method described here was also tested with several other subtropical and tropical plant tissues. The purity of the isolated small RNAs was sufficient for such applications as end-point stem-loop RT-PCR and northern blotting analysis, respectively. The simple and feasible extraction method reported here is expected to have excellent potential for isolation of small RNAs from recalcitrant plant tissues rich in polyphenols and polysaccharides.