Phenol-based Extraction Research Articles

Evaluation of three different DNA extraction methods for the detection of Pepper yellow leaf curl virus (PepYLCV) by Polymerase Chain Reaction

Abstract Pepper yellow leaf curl virus (PepYLCV) from the genus Begomovirus infecting Chili pepper plant can cause significant yield losses. PepYLCV infection can lead the development of mosaic yellow, mottled pattern and yellowing leaves, which can be challenging in disease management and control. Developing effective detection methods for PepYLCV is a critical disease management and crop loss mitigation. The Begomovirus viruses have extremely low viral concentrations and are limited to the phloem and vascular system. DNA extraction is an important step in PepYLCV detection. This study aimed to evaluate the efficiency of three DNA extraction methods: phenol-based extraction, CTAB-based extraction, and the GeneJET Plant Genomic DNA Purification Kit (Thermo Scientific™). These methods were evaluated for their performance to identified the presence of PepYLCV DNA and provide accurate results for further analyses. DNA samples were extracted from two varieties including Red Habanero and F8 012328-6-2-1-1-3-1, and were subsequently amplified using Krusty Homer’s primers. The results showed that all three extraction methods possess the capability to identify PepYLCV. Based on the results, DNA extraction employing the CTAB-based method yields DNA with higher concentration and purity levels. Additionally, this method proves to be cost-effective and proficient in generating higher DNA concentrations.

Improving gene expression analysis efficacy from formalin-fixed paraffin embedded tissues.

Improving RNA isolation and cDNA synthesis techniques has emerged due to advancements in the knowledge of molecular basis of most diseases. This in turn increased the need of higher quantity and quality of the extracted genetic material to be used for a variety of diagnostic tests and experiments. The aim of the study was to compare three modified methods for RNA extraction from formalin-fixed paraffin embedded (FFPE) biopsied tissue and different cDNA synthesis strategies to facilitate study of gene expression. Compared RNA extraction methods were: lysis buffer, phenol-based extraction, and combination of both with concomitant use of silica-based spin columns. RNA quantity and purity were estimated spectrophotometrically. Different priming strategies for cDNA synthesis were applied: oligodT, combination of oligodT and random hexamer, and gene specific primer. Two-step RT-qPCR of ribosomal protein L37A on preamplified and non-preamplified cDNA templates was performed. The combination of lysis buffer with phenol based extraction gave higher RNA yield. By doing cDNA preamplification, the confidence of detection by qPCR was raised, and efficiency was improved. The preamplified template increased the sensitivity of analysis. Together, the combination of approaches improved substantially the reproducibility and validity of quantitative gene expression analyses from FFPE tissues.

Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants.

Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer-based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta . The detergent-free phenol-based extraction combined with dodecyl-β-D-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groupsby LC-MS/MS. Additionally, dodecyl-β-D-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.

Effective protein extraction from mycelium and fruiting body of Auricularia auricula for proteomics studies

ABSTRACTAuricularia auricula is one of the most popular edible mushrooms in China. Limited information is available regarding genes and proteins related to A. auricula development from mycelium to mature fruiting body and response to environmental stimuli. With the development of proteomics, this technology platform for addressing this problem is available. A critical step of a good proteome is protein extraction which can gives the best quality and a wide coverage of total proteins. To establish an effective protein extraction protocol suitable for proteomics analysis in mycelium and fruiting body of A. auricula, four previously reported protein extraction protocols including direct extraction, trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation and phenol-based extraction, were compared based on the protein yield and 2-DE patterns. The efficiency of the four methods was evaluated by comparing protein yield and 2-DE patterns. Of the four methods, both TCA precipitation and phenol-based extraction method for A. auricula mycelia protein extraction yielded the good protein separation pattern and higher number of protein spots in 2-DE analysis. Furthermore, phenol-based extraction is superior to the other tested methods for A. auricula fruiting body proteome analysis .

Phenol-based Extraction of RNA from Chlamydomonas reinhardtii

Phenol-based Extraction of RNA from Chlamydomonas reinhardtii

Proteome profile of salt gland-rich epidermis extracted from a salt-tolerant tree species.

Preparation of proteins from salt-gland-rich tissues of mangrove plant is necessary for a systematic study of proteins involved in the plant's unique desalination mechanism. Extraction of high-quality proteins from the leaves of mangrove tree species, however, is difficult due to the presence of high levels of endogenous phenolic compounds. In our study, preparation of proteins from only a part of the leaf tissues (i.e. salt gland-rich epidermal layers) was required, rendering extraction even more challenging. By comparing several extraction methods, we developed a reliable procedure for obtaining proteins from salt gland-rich tissues of the mangrove species Avicennia officinalis. Protein extraction was markedly improved using a phenol-based extraction method. Greater resolution 1D protein gel profiles could be obtained. More promising proteome profiles could be obtained through 1D-LC-MS/MS. The number of proteins detected was twice as much as compared to TUTS extraction method. Focusing on proteins that were solely present in each extraction method, phenol-based extracts contained nearly ten times more proteins than those in the extracts without using phenol. The approach could thus be applied for downstream high-throughput proteomic analyses involving LC-MS/MS or equivalent. The proteomics data presented herein are available via ProteomeXchange with identifier PXD001691.

Evaluation of optimal RNA extraction method from human carotid atherosclerotic plaque.

Investigating molecular mechanisms involved in the formation of carotid atherosclerotic plaques has been challenging. Isolating high-quality RNA from plaque tissue can be difficult because of acellularity, calcification, and degradation. It is essential that the mRNA isolated from this tissue preserves and reflects the actual relative gene expression. Two common methods for RNA preservation, snap-freezing and stabilizing reagent, were compared using surgically resected human carotid atherosclerotic tissue. In addition, isolation methods were compared for integrity and quantity: column-based extraction, phenol-based extraction, and a combination of the two. We found that using a stabilizing reagent with column filtration resulted in the lowest yield and quality. Phenol-based extraction resulted in higher yields but also increased fragmentation. Snap-frozen tissue coupled with column-based extraction yielded the highest quality. The higher quality and quantity RNA obtained when processing snap-frozen tissue with column-based extraction make it possible to use difficult sample types for molecular downstream applications.

PROTEIN EXTRACTION OF MAIZE (ZEA MAYS) FOR PROTEOMIC 2 – DE ANALYSIS

A strength of two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is its ability to resolve and investigate the abundance of several thousand proteins in a single sample. Two different extraction procedures for two-dimensional electrophoresis of plant proteins are compared in this work. Phenol-based extraction methods have been mainly used to extract proteins from different organs or tissues on many species. We wanted to determine which of these protocols was optimal for starch plants in order to achieve both efficient protein extraction and high spot resolution on 2-D gels. The phenol-based protocol was superior to the sodium phosphatase methods, showing larger protein yields and greater spot resolution on 2-D gels.

An efficient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells

Firefly bioluminescence is widely used in the measurement of adenosine 5′-triphosphate (ATP) levels in biological materials. For such assays in tissues and cells, ATP must be extracted away from protein in the initial step and extraction efficacy is the main determinant of the assay accuracy. Extraction reagents recommended in the commercially available ATP assay kits are chaotropic reagents, trichloroacetic acid (TCA), perchloric acid (PCA), and ethylene glycol (EG), which extract nucleotides through protein precipitation and/or nucleotidase inactivation. We found that these reagents are particularly useful for measuring ATP levels in materials with relatively low protein concentrations such as blood cells, cultured cells, and bacteria. However, these methods are not suitable for ATP extraction from tissues with high protein concentrations, because some ATP may be co-precipitated with the insolubilized protein during homogenization and extraction, and it could also be precipitated by neutralization in the acid extracts. Here we found that a phenol-based extraction method markedly increased the ATP and other nucleotides extracted from tissues. In addition, phenol extraction does not require neutralization before the luciferin–luciferase assay step. ATP levels analyzed by luciferase assay in various tissues extracted by Tris–EDTA–saturated phenol (phenol–TE) were over 17.8-fold higher than those extracted by TCA and over 550-fold higher than those in EG extracts. Here we report a simple, rapid, and reliable phenol–TE extraction procedure for ATP measurement in tissues and cells by luciferase assay.

Cyanobacterial diversity for an anthropogenic impact assessment in the Sør Rondane Mountains area, Antarctica

AbstractThe recently inaugurated Belgian Princess Elisabeth Station has been built in the Sør Rondane Mountains (Dronning Maud Land). The construction site is situated close to the Utsteinen Nunatak (71°57′S, 23°20′E), on a granite ridge. Prior to the survey and construction activities, the site had been subject to limited or no direct human impacts. Therefore, a biological inventory has been started for future evaluations of human impacts in the area. This study presents the cyanobacterial diversity of ten samples from the Utsteinen ridge, the Utsteinen Nunatak in the vicinity of the station's site and the more distant Ketelersbreen Dry Valley. All samples were taken before the station was built and were studied by light microscopy and Denaturing Gradient Gel Electrophoresis. Two strains were also isolated. Two different phenol-based extraction methods, with and without detergent-based steps, were tested to improve the quality of molecular detection from these environmental samples. We observed high cyanobacterial diversity (ten morphotypes and 13 operational taxonomic units) in comparison to other Antarctic terrestrial locations and a widespread distribution pattern within the Sør Rondane Mountains area. This might reflect the habitat similarities and/or the easy local dispersal capabilities of microorganisms within the region. The high diversity and proportion of potential endemic (46%) operational taxonomic units, of which 23% were newly described, suggest that this area could have acted as a biological refuge during past glaciations.

Optimization of protein extraction and solubilization for mature grape berry clusters

Protein extraction from grape berries has been challenging, particularly in mature berries, which can have sugar concentrations as high as 26%. Grape skins and seeds contain large amounts of polyphenols, which can also interfere with efficient protein extraction. In plants, two extraction protocols, TCA/acetone-based and phenol-based methods, have been mainly used to extract proteins from different organs or tissues on many species. However, few results have been reported for grape berry clusters. We wanted to determine which of these protocols was optimal for berry clusters in order to achieve both efficient protein extraction and high spot resolution on 2-D gels. Four protocols, derived from either TCA/acetone or phenol procedures, were tested on mature Cabernet Sauvignon whole berry clusters. The phenol-based protocols were superior to the TCA/acetone methods, showing larger protein yields and greater spot resolution on 2-D gels. One method was clearly superior to the rest, a phenol-based extraction method combined with resuspension in the presence of both urea and thiourea as chaotropes. A total of 81 spots were excised and identified following MALDI-TOF/TOF MS analyses. Their identification helped further characterize the specificity of each extraction procedure.

Protein extraction from grape tissues by two-dimensional electrophoresis

At the onset of proteomic studies protein samples have to be accurately separated by two dimensional electrophoresis (2-DE); subsequently polypeptides are identified. Grape tissues, in particular roots, can be very problematic due to their hardness and to the high content of compounds that interfere in classical protein extraction. We have used a phenol-based extraction method in the presence of a protease inhibitor and Polyvinylpolypyrrolidone (PVPP). In this paper we demonstrate that this extraction method gives satisfactory and reproducible protein separation allowing the identification of some proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Serum, plasma and paraffin-embedded tissues as sources of DNA for studying cancer susceptibility genes.

The ability to isolate DNA from archived human serum, plasma and paraffin-embedded human tissues enhances opportunities to study breast, lung and other cancer risk factors. We report herein a simple and fast protocol for the extraction of genomic DNA from these sources. Using a phenol-based extraction method, the recovery for DNA is quantitative and reproducible. DNA yields in serum (250 microl) were between 162 and 1060 ng (n = 18 subjects), in plasma (250 microl) were between 165 and 375 ng (n = 5 subjects) and in embedded tissues (5-microm thick sections for ethanol fixed, and between 5- and 20-microm sections for formaldehyde fixation) were between 1 microg and 11.7 microg (n = 32 subjects). The extraction method was combined with newly designed PCR-based assays for cancer susceptibility marker genes such as CYP1A1 (exon 7), CYP2E1 (Dra1, Rsa1), GSTM1 and NAT2 [NAT2*5A (C481T), NAT2*6A (G590A), NAT2*7A (G857A)]. Genotyping results from the serum and paraffin-embedded tissues compared favorably to results from archived freshly frozen tissues, where concordance was 98% for serum, 100% for ethanol-fixed embedded tissues, and 97% for formaldehyde-fixed and paraffin-embedded tissues. This facile method will allow for the use of archived tissue samples of prospective cohort and other studies where intact DNA was not previously available.