Isolation of high quality RNA from soil-grown Ilex paraguariensis roots suitable for next-generation sequencing and gene expression analyses

Extraction of high quality RNA is a prerequisite for downstream application in functional genomics analyses. However, the extraction and purification of pure nucleic acids from root tissues is generally difficult due to the high concentration of carbohydrates and secondary metabolites. Furthermore, the presence of enzymatic inhibitors such as fulvic and humic acids can also negatively affect extraction quality, when extracting from clay soil-grown roots. In this work, total RNA was extracted from soil-grown roots of Ilex paraguariensis using four commercially available kits: SpectrumTM, RNeasy®, TRI Reagent®, and SV Total RNA Isolation System. Spectral measurement and electrophoresis were used to demonstrate RNA quality and quantity. The SpectrumTM and RNeasy® protocols provided the highest quantity and quality of RNA; however, the former revealed superior extraction performance. Consequently, total RNA was extracted from the roots of non-stressed and drought-stressed plants using the SpectrumTM method and six RNA-seq libraries were prepared from polyA + mRNAs by means of TruSeq mRNA library construction protocol to convert RNA to complementary DNA (cDNA). More than 80 million raw read sequences were obtained from each condition with an average read length of 150 bp. The yield and quality of the total RNA were consistently high and the RNA could be used for further analyses as demonstrated by cDNA library construction, RT-PCR, and transcriptome sequencing. Thus, SpectrumTM method can be used to isolate high quality RNA from roots of normal and drought stressed I. paraguariensis plants.Fil: Avico, Edgardo Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; ArgentinaFil: Acevedo, Raúl Maximiliano. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Departamento de Física y Química; ArgentinaFil: Calzadilla, Pablo Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ruiz, Oscar Adolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Sansberro, Pedro Alfonso. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Botánica del Nordeste. Universidad Nacional del Nordeste. Facultad de Ciencias Agrarias. Instituto de Botánica del Nordeste; Argentin

Modification of a commercial dna extraction kit to simultaneously recover rna, safely and rapidly, and to assess molecular biomass of the total and the active part of microbial communities, from soils with diverse mineralogy and carbon content : S11.04-P -15

We have modified a commercial DNA extraction kit for soil to simultaneously co-extract RNA. In this new procedure RNA and DNA are separated by two selective purifications in cascade without the need of DNAase or RNAse digestion. Consequently DNA and RNA are respectively purified from the whole co-extraction solution. Nucleic acids extraction is based on the action of SDS coupled with an efficient bead-beating step, but it does not require any solvent. Avoiding the use of solvents, which are damaging for human health and environmental quality, was one of our most important motivations to develop this protocol. In a second time, we have optimized this protocol to improve the DNA and RNA yield, but kipping those yields below the saturation limit of the kit to assess and quantify the variations of molecular biomass of the total (DNA) and the active (RNA) part of microbial communities in natural samples. We have also introduced a first step of homogenization of soil sample in liquid nitrogen to improve the reliability of the fungal 18S gene sequence quantification. Finally, we have shown that this protocol can be applied to a wide diversity of soils whatever their mineralogy and metal content (2 Ferralsols, 1 Vertisol, 2 Andosols from Madagascar), texture or biomass content (1 poor sandy soil from Congo and one carbon rich temperate soil sample submitted or not to a 1 month cold stress). * E Tournier, L. Amenc and AL. Pablo contributed equally to this study. (Texte intégral

Rapid plant DNA and RNA extraction protocol using a bench drill

Plant DNA and RNA extraction methods are well established, with a wide range of protocols, depending on the purposes of each laboratory/research. Nowadays, quick, inexpensive and easy plant DNA and RNA extraction methods are highly sought after. We developed an optimized protocol for plant DNA and RNA extraction that uses an inexpensive bench drill and plastic bags and does not require liquid nitrogen. DNA from leaves and RNA from leaves and roots of banana, pineapple, citrus, papaya, passion fruit and cassava, were extracted using a basic cetyltrimethylammonium bromide method. Both nucleic acids were quantified and evaluated for quality based on agarose gel electrophoresis. The DNA and RNA extractions were successful for all species, and RNA quality in pellets was maintained after storage at room temperature for three weeks. This protocol can reduce costs considerably in laboratories with ongoing routine activities of DNA and RNA extraction for genetic diversity and gene expression analyses, where other conventional methods have not been successful due to explant, condition of samples and quantity and quality of nucleic acids. This is especially relevant for many laboratories in developing countries where the cost and availability of liquid nitrogen may be a constraint

RNA extraction from self-assembling peptide hydrogels to allow qPCR analysis of encapsulated cells

Self-assembling peptide hydrogels offer a novel 3-dimensional platform for many applications in cell culture and tissue engineering but are not compatible with current methods of RNA isolation; owing to interactions between RNA and the biomaterial. This study investigates the use of two techniques based on two different basic extraction principles: solution-based extraction and direct solid-state binding of RNA respectively, to extract RNA from cells encapsulated in four β-sheet forming self-assembling peptide hydrogels with varying net positive charge. RNA-peptide fibril interactions, rather than RNA-peptide molecular complexing, were found to interfere with the extraction process resulting in low yields. A column-based approach relying on RNA-specific binding was shown to be more suited to extracting RNA with higher purity from these peptide hydrogels owing to its reliance on strong specific RNA binding interactions which compete directly with RNA-peptide fibril interactions. In order to reduce the amount of fibrils present and improve RNA yields a broad spectrum enzyme solution—pronase—was used to partially digest the hydrogels before RNA extraction. This pre-treatment was shown to significantly increase the yield of RNA extracted, allowing downstream RT-qPCR to be performed

Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

Background: Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). Methods: Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. Results: Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. Conclusions: Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material

A High-Throughput Method for Illumina RNA-Seq Library Preparation.

With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation

Rapid bedside inactivation of Ebola virus for safe nucleic acid tests

Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available MagNA Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding MagNA Pure lysis/binding buffer directly into vacuum blood collection EDTA-tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months and Ebola virus RNA is preserved in the MagNA Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from MagNA Pure lysis/binding buffer-inactivated samples using the QIAamp Viral RNA mini kit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection

Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

The success of fundamental and applied nucleic acid (NA) research depends on NA purity, but obtaining pure NAs from raw, unprocessed samples is challenging. Purification using solid-phase NA extractions utilizes sequential additions of lysis and wash buffers followed by elution. The resulting eluent contains NAs and carryover of extraction buffers. Typically, these inhibitory buffers are heavily diluted by the reaction mix (e.g., 10x dilution is 1 µL eluent in 9 µL reaction mix), but in applications requiring high sensitivity (e.g., single-cell sequencing, pathogen diagnostics) it is desirable to use low dilutions (e.g., 2x) to maximize NA concentration. Here, we demonstrate pervasive carryover of inhibitory buffers into eluent when several commercial sample-preparation kits are used following manufacturer protocols. At low eluent dilution (2–2.5x) we observed significant reaction inhibition of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and reverse transcription (RT). We developed a two-phase wash (TPW) method by adding a wash buffer with low water solubility prior to the elution step. The TPW reduces carryover of extraction buffers, phase-separates from the eluent, and does not reduce NA yield (measured by digital PCR). We validated the TPW for silica columns and magnetic beads by demonstrating significant improvements in performance and reproducibility of qPCR, LAMP, and RT reactions

cDNA-RNA subtractive hybridization reveals increased expression of mycocerosic acid synthase in intracellular Mycobacterium bovis BCG.

Identifying genes that are differentially expressed by Mycobacterium bovis BCG after phagocytosis by macrophages will facilitate the understanding of the molecular mechanisms of host cell-intracellular pathogen interactions. To identify such genes a cDNA-total RNA subtractive hybridization strategy has been used that circumvents the problems both of limited availability of bacterial RNA from models of infection and the high rRNA backgrounds in total bacterial RNA. The subtraction products were used to screen a high-density gridded Mycobacterium tuberculosis genomic library. Sequence data were obtained from 19 differential clones, five of which contained overlapping sequences for the gene encoding mycocerosic acid synthase (mas). Mas is an enzyme involved in the synthesis of multi-methylated long-chain fatty acids that are part of phthiocerol dimycocerosate, a major component of the complex mycobacterial cell wall. Northern blotting and primer extension data confirmed up-regulation of mas in intracellular mycobacteria and also revealed a putative extended -10 promoter structure and a long untranslated upstream region 5' of the mas transcripts, containing predicted double-stranded structures. Furthermore, clones containing overlapping sequences for furB, groEL-2, rplE and fadD28 were identified and the up-regulation of these genes was confirmed by Northern blot analysis. The cDNA-RNA subtractive hybridization enrichment and high density gridded library screening, combined with selective extraction of bacterial mRNA represents a valuable approach to the identification of genes expressed during intra-macrophage residence for bacteria such as M. bovis BCG and the pathogenic mycobacterium, M. tuberculosis

Investigation into the relationship between ethylene and sulfur assimilation in Arabidopsis thaliana and onion (Allium cepa L.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (with Honours) in Biochemistry at Massey University

The phytohormone ethylene (C2H4) mediates the adaptive responses of plants to various nutrient deficiencies including iron (Fe)-deficiency, phosphorus (P)-deficiency and potassium (K)-deficiency. However, evidence for the involvement this hormone in the sulfur (S) deficiency response is limited to date. In this study, the effect of C2H4 treatment on the accumulation of the S-assimilation enzymes ATP sulfurylase (ATPS). adenosine-5 -phosphosulfate-reductase (APR), O-acetylserine-(thiol)-lyase (OASTL) and sulfite reductase (SiR) was examined in A. thaliana and onion (A. cepa). To complement this, the effect of short-term S-depletion on the expression of the 12-member gene family of the C2H4 biosynthetic enzyme, l-amino-cyclopropane-l-carboxylic acid (ACC) synthase (ACS) from A. thaliana, designated AtACS1-12, was also examined. Western analyses were used to show that plants of A. thaliana pre-treated with the C2H4-signalling inhibitor 1-MCP, had elevated levels of ATPS, APR and OASTL protein in leaf tissue at all time points examined, suggesting that C2H4 has an inhibitory effect on the accumulation of these enzymes. However, SiR appeared to be under dual regulation by C2H4: under S-sufficient conditions C2H4 appears to prevent the unnecessary accumulation of SiR and conversely promote the fast accumulation of SiR under S-depleted conditions. The changes in AtACS1-12 expression in the root and leaf tissues of S-sufficient and S-depleted plants of A. thaliana were examined by RT-PCR using gene-specific, exon-spanning primers. The expression patterns of AtACS2, AtACS6 and AtACS7 were comparable regardless of S availability and may therefore be housekeeping genes. In contrast, the expression of AtACS5 in leaf, and AtACS8 and AtACS9 in roots was repressed under S-depleted conditions, although the mechanism of this repression cannot be elucidated from this study. The protein products of these closely-related genes are believed to be phosphorylated and stabilised by a CDPK whose activity may be compromised by S-depletion. The inhibition of AtACS5, AtACS8 and AtACS9 expression, and the decrease in AtACS5, AtACS8 and AtACS9 accumulation, and hence less C2H4 production, may be part of the plant adaptive response to S-depletion, as the C2H4 -mediated repression of root growth is alleviated to allow the plant to better seek out the lacking nutrient. The expression of the MPK-stabilised genes AtACS2 and AtACS6 appeared to be similar regardless of S availability, although this may merely be a consequence of the scoring method used in this study, which cannot determine whether there was any difference in the level of expression of these genes. The expression of AtACS10 and AtACS12 was repressed in S-deficient plants. Although both AtACS10 and AtACS12 isozymes posses the hallmark seven conserved regions found in the ACSes of other plant species, they are also phylogenetically related to alanine and aspartate aminotransferases, and are known to encode aspartate (AtACS10) and aromatic amino acid transaminases (AtACS12). Therefore, the apparent downregulation of these genes suggests that the downregulation of amino acid metabolism may be part of the plant adaptive response to S-depletion. The downregulation of several AtACS genes, and therefore possibly also C2H4 biosynthesis, in S-deficient plants was accompanied by an accumulation of APR protein. The increase in APR protein that also occurred in 1-MCP-treated plants indicates that C2H4 may be involved in the plant response to S-depletion, because in both cases the upregulation of the S-assimilation pathway, as manifested by the accumulation of APR protein, occurred when C2H4 biosynthesis and signalling was repressed. However, the possible role of other phytohormoes in the plant response to S-depletion cannot be excluded, as there is evidence for crosstalk between the C2H4 signalling pathway and those of auxin, abscisic acid (ABA), cytokinins and jasmonic acid (JA). Furthermore, because C2H4 has been implicated in the response of various plants to Fe-deficiency, P-deficiency, and K-deficiency, in addition to S-deficiency, it may be a regulator of the plant adaptive response to nutrient stresses in general