scholarly journals Protocol adjustment improves the extraction of high-quality total RNA from common bean stems infected by Sclerotinia sclerotiorum

2019 ◽  
Vol 43 ◽  
Author(s):  
Rafael Novais de Miranda ◽  
Caroline Marcela da Silva ◽  
Antonio Carlos da Mota Porto ◽  
Welison Andrade Pereira
Keyword(s):  
Gene Expression ◽  
Common Bean ◽  
Sclerotinia Sclerotiorum ◽  
Rna Extraction ◽  
White Mold ◽  
Basic Requirement ◽  
High Quality ◽  
Commercial Kits ◽  
Made In

ABSTRACT The Straw Test is an assay developed to evaluate the resistance of common bean to white mold, in which the plant stems are inoculated and the symptoms of the disease are monitored. It is plausible to admit that investigating gene expression in pathogen-infected tissues may be strategically interesting. However, obtaining a quality RNA is a basic requirement for this purpose. Therefore, the objective of this study was to evaluate adjustments in protocols of commercial kits in the expectation of improving the quality of RNA obtained from bean stems. For this, plants of two lines were inoculated and the stems pathogen-infected were collected 72 hours after. For RNA extraction, two commercial reagents were used following the manufacturer’s recommendations and then following adaptations in these protocols. In particular, the proposed modifications relate to volumes of supernatant recovered in purification steps, additional step of chloroform purification and extended time for nucleic acids precipitation. The obtained RNA was analyzed by spectrophotometer, electrophoresis and bioanalyzer, then converted into cDNA and subsequently submitted to PCR. From the obtained data, it was observed that the adaptations made in the protocols contributed to better results and that, when the indicative values of RNA quality are guaranteed, the subsequent reactions are more pure, precise and representative.

scholarly journals The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽  
2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Michal Marczyk ◽  
Chunxiao Fu ◽  
Rosanna Lau ◽  
Lili Du ◽  
Alexander J. Trevarton ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Rna Sequencing ◽  
Rna Extraction ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
The Impact ◽  
Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

Efficacy of three greenhouse screening methods for the identification of physiological resistance to white mold in dry bean

2009 ◽  
Vol 89 (4) ◽  
pp. 755-762 ◽  
Author(s):  
H Terán ◽  
S P Singh
Keyword(s):  
Phaseolus Vulgaris ◽  
Common Bean ◽  
Sclerotinia Sclerotiorum ◽  
Screening Method ◽  
White Mold ◽  
Screening Methods ◽  
Post Inoculation ◽  
Dry Bean ◽  
Physiological Resistance ◽  
Nueva Granada

White mold (WM) caused by Sclerotinia sclerotiorum (Lib.) de Bary is the most devastating disease of common bean (dry and snap or garden bean) (Phaseolus vulgaris L.) in North America. The use of a reliable screening method (SM) in common bean is crucial to improve physiological resistance to WM. The objective of this study was to compare the efficacy of three SM to identify physiological resistance in dry bean genotypes with different evolutionary origins and levels of resistance. Screening methods tested were: (i) the modified straw test or cut–stem (CSM); (ii) infected bean flower (IFL); and (iii) infected oat seed (IOS). A 195, ICA Bunsi, Othello, and VCW 54 dry bean were tested with the three SM. The experimental design was a split plot in randomized complete blocks with three replications in 2007 and 2008. Two independent inoculations 1 wk apart for each SM were made. The WM reaction was scored at 16, 23, and 33 d post-inoculation (DPI) using a 1 to 9 scale. There were highly significant differences between SM and its interaction with years. The CSM and IFL were the most consistent and highly correlated (r > 0.70, P < 0.01). Interspecific breeding line VCW 54 consistently had the highest WM resistance across years, SM, and evaluation dates, followed by A 195. White mold scores increased with delayed evaluations. Thus, CSM or IFL with disease assessed 33 DPI should be used for identifying common bean genotypes with high levels of physiological resistance to WM.Key words: Common bean, growth habit, race Mesoamerica, race Nueva Granada, Phaseolus vulgaris, Sclerotinia sclerotiorum

Isolation of high-quality total RNA from rumen anaerobic bacteria and fungi, and subsequent detection of glycoside hydrolases

2011 ◽  
Vol 57 (7) ◽  
pp. 590-598 ◽  
Author(s):  
Pan Wang ◽  
Meng Qi ◽  
Perry Barboza ◽  
Mary Beth Leigh ◽  
Emilio Ungerfeld ◽  
...  
Keyword(s):  
Gene Expression ◽  
Solid Phase ◽  
Large Subunit ◽  
Rna Extraction ◽  
Small Subunit ◽  
Glycoside Hydrolases ◽  
Small Subunit Rrna ◽  
High Quality ◽  
Major Barrier ◽  
Total Rna

The rumen is one of the most powerful fibrolytic fermentation systems known. Gene expression analyses, such as reverse transcription PCR (RT-PCR), microarrays, and metatranscriptomics, are techniques that could significantly expand our understanding of this ecosystem. The ability to isolate and stabilize representative RNA samples is critical to obtaining reliable results with these procedures. In this study, we successfully isolated high-quality total RNA from the solid phase of ruminal contents by using an improved RNA extraction method. This method is based on liquid nitrogen grinding of whole ruminal solids without microbial detachment and acid guanidinium – phenol – chloroform extraction combined with column purification. Yields of total RNA were as high as 150 µg per g of fresh ruminal content. The typical large subunit/small subunit rRNA ratio ranged from 1.8 to 2.0 with an RNA integrity number (Agilent Technologies) greater than 8.5. By eliminating the detachment step, the resulting RNA was more representative of the complete ecosystem. Our improved method removed a major barrier limiting analysis of rumen microbial function from a gene expression perspective. The polyA-tailed eukaryotic mRNAs obtained have successfully been applied to next-generation sequencing, and metatranscriptomic analysis of the solid fraction of rumen contents revealed abundant sequences related to rumen fungi.

High quality RNA from hydroponically grown grapevine roots suitable for gene expression studies

2017 ◽  
Vol 42 (4) ◽  
Author(s):  
Synda Chenenaoui ◽  
Samia Daldoul ◽  
Ahmed Mliki
Keyword(s):  
Gene Expression ◽  
Rna Extraction ◽  
Extraction Procedure ◽  
Rna Isolation ◽  
Extraction Methods ◽  
Purification Step ◽  
High Quality ◽  
Rna Quality ◽  
Expression Studies ◽  
Gene Expression Studies

AbstractObjectives:Grapevine root system plays a great role in sensing and adapting to abiotic and biotic stresses. Identification of candidate genes involved in the tolerance to abiotic stress is becoming a crucial strategy to select and breed resilient genotypes. However, obtaining high quality RNA from grapevine roots under hydroponic culture is difficult. Hence, we have developed a new extraction procedure to improve RNA quality for root gene expression studies.Methods:Conventional RNA extraction methods using CTAB are not suitable for gene expression studies and need to be improved. Here we report the application of a CTAB- based method for RNA extraction using an additional clean-up purification step.Results:The RIN value of the resulting RNA indicated that our procedure allowed the purification of high RNA quality and quantity. Hence, the clean-up purification step efficiently eliminated contaminants which inhibit downstream applications. Derived RNA was successfully used for differential gene expression analysis in salt stressed grapevine by Northern Blot hybridizations.Conclusion:In this study, we developed an efficient RNA isolation protocol from hydroponic cultivated grapevine roots which yielded RNA suitable for gene expression studies. This will open large perspectives in grapevine functional genomics with the identification of pertinent genes of agronomic interest.

scholarly journals RESULTS OF TREATMENT OF EDENTULOUS PATIENTS WITH DENTURES MADE OF «FTORAX»

2020 ◽  
pp. 37-45
Author(s):  
V.M. Dvornyk ◽  
H.M. Kuz ◽  
O.B. Tumakova ◽  
O.S. Shemetov ◽  
V.S. Kuz
Keyword(s):  
Morphological Changes ◽  
Base Material ◽  
Masticatory Muscles ◽  
High Quality ◽  
Results Of Treatment ◽  
Acrylic Copolymers ◽  
Personal Approach ◽  
Edentulous Patients ◽  
Made In

Background. The problem of providing high-quality dental care to edentulous patients remains one of the most important and unresolved to this day in the clinic of prosthodontics. The causes of complete loss of teeth can be both periodontal tissue diseases and diseases of hard tooth tissues of carious and non-carious origin, trauma and the like. It becomes necessary to fabricate complete removable dentures to prevent the occurrence of pathologies in such situations. Complete secondary adentia leads to a large number of local and general complications. The chewing apparatus undergoes a number of functional and morphological changes. Therefore, the complete absence of teeth sets the dentist the task of restoring the functions of full chewing, the appearance of the patient, and speech. The complexity of high-quality prosthetics for edentulous patients is also because the clinical characteristics of supporting tissues are diverse, constantly changing, so a personal approach to creating stable denture is needed. It should be added that the fabrication of high-quality complete removable dentures largely depends on the properties of the base material. The main group of materials for the fabrication of such dentures is acrylic plastics. The aim. The work is focused on the study of the functional state of the masticatory muscles and the restoration of masticatory effectiveness in edentulous patients, for whom dentures were made in the prosthodontics clinic from the acrylic base material «Ftorax». Material and methods. Hot curing plastic based on fluorine-containing acrylic copolymers «Ftorax» was used in the work. Our evaluation of the quality of all constructions was carried out using the «BOFSAS» test, determining the biopotentials of the masticatory muscles using electromyography, and determining the masticatory effectiveness according to I.S. Rubinov. Results. Based on our work, it can be noted that the use of the basic acrylic material «Ftorax» allows us to achieve good fixation and stabilization of complete removable dentures, which is subjectively confirmed by the «BOFSAS» test, objectively – by the electromyographic studies and time indices during chewing test according to I.S. Rubinov. The amplitude during volitional compression is 556.90±8.72 μV, which approaches the norm (641.58±10.01 μV), and almost four times higher than before prosthetics (188.11±8,13 μV) after 1 year of constructions using. A similar pattern is observed with arbitrary chewing: the amplitude (547.32±8.43 μV) approaches normal (643.92±9.11 μV) compared with the results before prosthetics (201.40±9.39 μV). As for the coefficient «K», its value decreased almost twofold compared with the results before prosthetics (2.44±0.14) and equals to 1.25±0.03, which is significantly closer to normal (1,02±0.01). After the test by Rubinov the following results were obtained: the average time that patients with intact dentitions spent chewing on the stimulus was approximately 12.97±0.13 seconds and the average time for patients before prosthetics (with old dentures) was approximately 45.16±0,41 sec. The time that patients spent chewing a nut after 1 month was 28.97±0.42 seconds, after 6 months – 26.94±0.44 seconds, and after 1 year of using the entures – 25.48±0.45 sec. Based on this, it can be summarized that adaptation to such dentures takes place at the appropriate time with minor corrections. Conclusion. The results of our clinical study indicate the feasibility of using «Ftorax» in prosthodontics clinic for treatment of edentulous patients.

scholarly journals 212 GENE EXPRESSION PROFILE OF PROTAMINES AND TRANSITION NUCLEAR PROTEINS IN BOVINE TESTIS

2013 ◽  
Vol 25 (1) ◽  
pp. 254 ◽  
Author(s):  
M. A. M. M. Ferraz ◽  
R. Simões ◽  
M. P. Milazzotto ◽  
J. A. Visintin ◽  
M. E. O. A. Assumpçäo
Keyword(s):  
Gene Expression ◽  
Rna Extraction ◽  
Great Influence ◽  
Software Tool ◽  
Nuclear Proteins ◽  
Dna Integrity ◽  
Evolutionary Mechanisms ◽  
Relative Expression ◽  
Bovine Sperm ◽  
Commercial Kits

During spermiogenesis, haploid spermatids undergo complex morphological and physiological changes to differentiate into spermatozoa. These processes include chromatin remodelling mediated by the replacement of histones through transition nuclear proteins (Tnp) and protamines (PRM). These proteins have the function to compact and protect the chromatin, exerting great influence on human and mouse fertility. Several studies have demonstrated the positive relationship between unregulated production of protamines and infertility. In humans and mice, PRM1/PRM2 ratio is important to predict fertility. When the 1 : 1 ratio (ideal) is disrupted, in these species, the sperm DNA integrity is altered. Most of the infertility cases caused by protamine deficiency, in humans, are related to PRM2 (Aoki et al. 2005 Hum. Reprod. 20, 1298–1306). Expression levels of PRM2 have been correlated with the DNA damage in mice; however, its role on bull fertility is still unclear. The aim of this study was to determine the relative expression of PRM1, PRM2, PRM3, Tnp1, and Tnp2 in bovine testis. Evaluate the expression of these genes is of utmost importance to understand the role of each protamine during bull spermatogenesis. Testis from post-pubertal bulls (n = 10) were obtained from the slaughterhouse. The RNA extraction and cDNA synthesis were performed using commercial kits. The gene expression (P1, P2, P3, Tnp1, and Tnp2) was determined by real-time RT-PCR using bovine specific primers and β-actin as endogenous controls. A relative expression software tool (Pfaffl et al. 2002 Nucleic Acid Res. 30) was used to compare all samples of each group. The quantification of mRNA relative expression demonstrated a higher expression of PRM1, the relative expression of PRM2 was lower than the relative expression of PRM1 (5.008 ± 1.501 × 23.906 ± 6.174, respectively; P < 0.05). There was no difference between the relative expression of the mRNA for PRM2, Tnp1, and Tnp2 (5.008 ± 1.501, 5.023 ± 1.064, 4.266 ± 1.170, respectively; P > 0.05). The PRM3 mRNA had the lowest relative expression (2.003 ± 0.663). The PRM1/PRM2 ratio found in this study was 4.77 : 1.00. Differently from human and mice, the lower expression of PRM2 mRNA may be an evolutionary adaptation of the bull spermatogenesis, which makes the bovine sperm less susceptible to protamination changes that lead to infertility. More studies are being performed by our research group to evaluate the function of these proteins in bulls. It is fundamental to understand the biology of bovine spermiogenesis, providing knowledge to increase the fertility and be able to elucidate the evolutionary mechanisms that may have caused the possible loss of functionality of PRM2 in bulls. This work was supported financially by FAPESP (09/17035-6 and 07/55294-8).

scholarly journals Screening common bean germplasm for resistance to genetically diverse Sclerotinia sclerotiorum isolates from Argentina

2020 ◽  
Vol 42 ◽  
pp. e42786
Author(s):  
Carla Luciana Abán ◽  
Gisel Maria Taboada ◽  
Norma Beatriz Casalderrey ◽  
Maria Elisa Maggio ◽  
Mario Osvaldo Chocobar ◽  
...  
Keyword(s):  
Common Bean ◽  
Sclerotinia Sclerotiorum ◽  
Durable Resistance ◽  
Weather Conditions ◽  
White Mold ◽  
Post Inoculation ◽  
Progress Curve ◽  
Breeding Programmes ◽  
Favorable Weather ◽  
Production Areas

White mold caused by Sclerotinia sclerotiorum (Lib.) de Bary is a devastating disease that affects the common bean (Phaseolus vulgaris. L) crop worldwide. In Argentina, white mold has been detected in all bean production areas, reaching seed yield and quality losses up to 100% on susceptible common bean cultivars under favorable weather conditions. The aim of this study was to screen the physiological resistance of 20 common bean accessions to five genetically distinct isolates of S. sclerotiorum collected from the main common bean growing area of Argentina, using the greenhouse straw test. The white mold reaction was scored at 7, 14, and 21 days post-inoculation using a 1 (no disease symptoms) to 9 (severely diseased or dead plants) scale and the area under the disease progress curve (AUDPC) was determined. Highly significant differences (p < 0.001) were observed between isolates, accessions and genotype x isolate interaction at the three evaluations dates. All cultivars and lines were susceptible at the end of the assessment, except line A 195 which was resistant to white mold against the five isolates tested and was significantly different from all accessions. This work represents a valuable contribution to regional breeding programmes aimed to obtain cultivars with durable resistance.

scholarly journals A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Amaranatha R. Vennapusa ◽  
Impa M. Somayanda ◽  
Colleen J. Doherty ◽  
S. V. Krishna Jagadish
Keyword(s):  
Rna Extraction ◽  
Poor Quality ◽  
High Yield ◽  
Plant Tissues ◽  
Universal Method ◽  
High Quality ◽  
Yield And Quality ◽  
Extraction Buffer ◽  
Co Precipitation

Abstract Using existing protocols, RNA extracted from seeds rich in starch often results in poor quality RNA, making it inappropriate for downstream applications. Though some methods are proposed for extracting RNA from plant tissue rich in starch and other polysaccharides, they invariably yield less and poor quality RNA. In order to obtain high yield and quality RNA from seeds and other plant tissues including roots a modified SDS-LiCl method was compared with existing methods, including TRIZOL kit (Invitrogen), Plant RNeasy mini kit (Qiagen), Furtado (2014) method, and CTAB-LiCl method. Modifications in the extraction buffer and solutions used for RNA precipitation resulted in a robust method for extracting RNA in seeds and roots, where extracting quality RNA is challenging. The modified SDS-LiCl method revealed intense RNA bands through gel electrophoresis and a nanodrop spectrophotometer detected ratios of ≥ 2 and 1.8 for A260/A230 and A260/A280, respectively. The absence of starch co-precipitation during RNA extraction resulted in enhanced yield and quality of RNA with RIN values of 7–9, quantified using a bioanalyzer. The high-quality RNA obtained was demonstrated to be suitable for downstream applications, such as cDNA synthesis, gene amplification, and RT-qPCR. The method was also effective in extracting RNA from seeds of other cereals including field-grown sorghum and corn. The modified SDS-LiCl method is a robust and highly reproducible RNA extraction method for plant tissues rich in starch and other secondary metabolites. The modified SDS-LiCl method successfully extracted high yield and quality RNA from mature, developing, and germinated seeds, leaves, and roots exposed to different abiotic stresses.

Bacillus sp. FSQ1: a Promising Biological Control Agent Against Sclerotinia sclerotiorum, the Causal Agent of white Mold in Common Bean (Phaseolus vulgaris L.)

2021 ◽  
Vol 48 (6) ◽  
pp. 729-739
Author(s):  
María Fernanda Villarreal-Delgado ◽  
Fannie Isela Parra-Cota ◽  
Luis Alberto Cira-Chávez ◽  
María Isabel Estrada-Alvarado ◽  
Sergio de los Santos-Villalobos
Keyword(s):  
Biological Control ◽  
Phaseolus Vulgaris ◽  
Common Bean ◽  
Sclerotinia Sclerotiorum ◽  
Biological Control Agent ◽  
Control Agent ◽  
Causal Agent ◽  
White Mold ◽  
Bacillus Sp ◽  
Phaseolus Vulgaris L

scholarly journals Performance of common bean seeds infected by the fungus Sclerotinia sclerotiorum

2013 ◽  
Vol 35 (2) ◽  
pp. 153-160 ◽  
Author(s):  
Luana da Silva Botelho ◽  
Willian Luis Antonio Zancan ◽  
José da Cruz Machado ◽  
Ellen Noly Barrocas
Keyword(s):  
Common Bean ◽  
Sclerotinia Sclerotiorum ◽  
Dry Weight ◽  
Dry Mass ◽  
White Mold ◽  
Inoculum Potential ◽  
Initial Inoculum ◽  
Aerial Parts ◽  
Productive Potential ◽  
Different Strains

The fungus Sclerotinia sclerotiorum, causal agent of white mold disease on several economically important crops, such as bean, soybean, and cotton, is commonly disseminated through seeds and can cause high losses on their quality and in productivity of these species. The aim of this study was assessing the effects of different initial inoculums potentials of this fungus on common bean seeds using two different strains of the fungus and two genotypes of common beans (Pérola and Ouro Negro) artificially inoculated. Seeds were sown on soil and the cultivation was performed under controlled environmental conditions favorable to development of the white mold disease. Variables assessed were: germination; seed health; emergence speed index; initial and final seedling number; and dry weight of aerial parts and roots. It was found that with the gradual increase in inoculum potential in the seeds also occurred gradual reduction in the values of: germination; emergence speed index; seedling stand; and length and dry mass of aerial parts and roots. These results show the importance of initial inoculum potential of S. sclerotiorum present in common bean seeds, as much in disseminating the pathogen as on direct damages caused in field by reducing productive potential of the emerged plants.

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