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.     

A Rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants

Introduction –  RNA quality and integrity are critical for many studies in plant molecular biology. High‐quality RNA extraction from grapevine and other woody plants is problematic due to the presence of polysaccharides, polyphenolics and other compounds that bind or co‐precipitate with the RNA. Objective  – To develop an optimised cetyltrimethylammonium bromide (CTAB)‐based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from polysaccharide‐rich tissues of several plants. Methodology  – Several changes were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C, the sample weight was decreased and the concentrations of PVP‐40 and LiCl were increased reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with six different RNA extraction methods from three grapevine tissues, namely, in vitro plantlets, and leaves and mature canes from actively growing field vines. Results –  The rapid CTAB method gave high‐quality RNA in only 3 h at low cost with efficiency equal to or higher than that obtained with other time‐consuming and expensive protocols. The procedure was applied to RNA extraction from other grapevine tissues and other woody species including olive, lemon, poplar, chestnut, apple, pear, peach, cherry, apricot, plum and kiwi fruit. RNA of high quality could be isolated from all tissues and from all species. Conclusion –  The study has shown that the improvement of a CTAB‐based protocol allows the rapid isolation of high‐quality RNA from grapevine and many woody species. Copyright © 2008 John Wiley & Sons, Ltd.     

A method for extracting genomic DNA suitable for medium-throughput applications from plant tissues rich in contaminants

Isolating nucleic acids from sources rich in contaminants is particularly cumbersome when treating a large number of samples. Several protocols have been published that address the problem of nucleic acid extraction and purification, but few address sample number. We describe a method for extracting DNA from recalcitrant tree species by using a commercial grinding apparatus. This alleviates the hard work of sample preparation prior to lysis and purification. Our method has been tested extensively on different fruit tree species and in projects that require the simultaneous processing of hundreds of samples. Moreover, it does not require the availability of robotic workstations.     

A bioassay for insect deterrent compounds found in plant and animal tissues

A general field bioassay for detecting biologically active compounds in plants and insects has been developed and tested for efficacy and sensitivity. Methanolic extracts, in sucrose solution, of 20 plant and six caterpillar species were offered to the ponerine ant Paraponera clavata and the feeding preferences observed. The bioassay resulted in the detection of nine plant and three caterpillar species with ant-deterrent extracts, and 11 plant and three caterpillar species with neutral or attractant extracts. All of the plants showing ant-deterrent characteristics which had been chemically investigated in our laboratory, or for which chemical literature was available, contained secondary metabolites of known deterrence. Both naturally occurring and artificial differences in chemical concentrations could be detected using the bioassay. The method provides a means of screening plants and insects for compounds that are insect anti-feedants or that can modify insect behaviour.     

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 universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq

Background

Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template.

Results

The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately flanking the poly(C) region.

Conclusions

We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-828) contains supplementary material, which is available to authorized users.     

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.