scholarly journals Efficient Method for Isolation of High-Quality RNA from Psidium Guajava L. Tissues

2020 ◽  
Author(s):  
Paola Avelar Carpinetti ◽  
Vinicius Sartori Fioresi ◽  
Thais Ignez Cruz ◽  
Francine Alves Nogueira Almeida ◽  
Drielli Canal ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Rna Extraction ◽  
Plant Molecular Biology ◽  
Rna Isolation ◽  
Psidium Guajava ◽  
Pcr Analysis ◽  
Flower Bud ◽  
High Quality ◽  
Rna Integrity ◽  
Lysis Buffer

Abstract Background: Acquiring high-quality RNA in sufficient amounts is necessary in plant molecular biology and genetic studies. Several methods of RNA extraction from plants are available in the literature, mainly due to the great biochemical diversity present in each species and tissue, which can complicate or prevent the extraction. Psidium guajava (Myrtaceae family) is a perennial fruit tree of medicinal and economic value; nevertheless, only few molecular and omics studies are available for the species. One reason for this fact is the difficulty in obtaining the RNA due to the content of the samples, which are rich in polyphenols, polysaccharides and secondary metabolites. Furthermore, there is still no tested or standardized method available for the isolation of RNA from guava or Psidium samples, which hampers advances in the genus.Results: Here we compare the quality and yields of RNA isolated using six extraction protocols: two based on the application of cetyltrimethylammonium bromide (CTAB) lysis buffer, two commercial kits (PureLink™ RNA Mini Kit and RNeasy® Plant Mini Kit), one using the TRIzol® reagent, and one applying guanidine thiocyanate lysis buffer followed by organic phase extraction. RNA integrity, quality and yields were assessed by agarose gel electrophoresis and spectrophotometry. The CTAB-based method provided the highest RNA yields and quality for five different tissues (flower bud, immature leaf, young leaf, mature leaf and root), genotypes and stress conditions. For the most efficient protocol, the average yield of RNA from guava leaf was 210.4 μg/g of tissue, and the A260/A280 and A260/A230 ratios were 2.1 and 2.2, respectively. RT-PCR analysis demonstrated that the purity of the samples was sufficient for molecular biology experiments.Conclusion: CTAB-based methods for RNA isolation were found to be the most efficient, providing the highest RNA yields and quality for tissues from P. guajava. Additionally, they demonstrated to be compatible for downstream RNA-based applications, besides showing the advantages of lower cost and time investments.

scholarly journals Efficient method for isolation of high-quality RNA from Psidium guajava L. tissues

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0255245
Author(s):  
Paola de Avelar Carpinetti ◽  
Vinicius Sartori Fioresi ◽  
Thais Ignez da Cruz ◽  
Francine Alves Nogueira de Almeida ◽  
Drielli Canal ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Economic Value ◽  
Rna Extraction ◽  
Plant Molecular Biology ◽  
Rna Isolation ◽  
Cost Effective ◽  
Psidium Guajava ◽  
Flower Bud ◽  
High Quality ◽  
Lysis Buffer

Acquiring high-quality RNA in sufficient amounts is crucial in plant molecular biology and genetic studies. Several methods for RNA extraction from plants are available in the literature, mainly due to the great biochemical diversity present in each species and tissue, which can complicate or prevent the extraction. Psidium guajava (Myrtaceae family) is a perennial fruit tree of medicinal and economic value; nevertheless, only a few molecular studies are available for the species. One reason is the difficulty in obtaining RNA due to the content of the samples, which are rich in polyphenols, polysaccharides, and secondary metabolites. Furthermore, there are few studies available for the isolation of RNA from guava or Psidium samples, which hampers advances in the study of the genus. Here, quality and yields of RNA isolates were compared using six extraction protocols: two protocols based on the application of cetyltrimethylammonium bromide (CTAB) lysis buffer, one protocol which uses the TRIzol reagent, one which applies guanidine thiocyanate lysis buffer followed by organic phase extraction, and two commercial kits (PureLink RNA Mini Kit and RNeasy Plant Mini Kit). The CTAB-based method provided the highest RNA yields and quality for five different tissues (flower bud, immature leaf, young leaf, mature leaf, and root), genotypes, and stress conditions. For the most efficient protocol, the average yield of RNA from guava leaves was 203.06 μg/g of tissue, and the A260/A280 and A260/A230 ratios were 2.1 and 2.2, respectively. RT-qPCR analysis demonstrated that the purity of the samples was sufficient for molecular biology experiments. CTAB-based methods for RNA isolation were found to be the most efficient, providing the highest RNA yields and quality for tissues from P. guajava. Additionally, they were compatible for downstream RNA-based applications, besides being simple and cost-effective.

scholarly journals Modified CTAB protocol for RNA extraction from Lemon balm (Melissa officinalis L.)

2020 ◽  
Vol 115 (1) ◽  
pp. 53
Author(s):  
Fatemeh RAHMANI ◽  
Leila AMRAEE
Keyword(s):  
Molecular Biology ◽  
Secondary Metabolites ◽  
Ribonucleic Acid ◽  
Rna Extraction ◽  
Plant Molecular Biology ◽  
Rt Pcr ◽  
Melissa Officinalis ◽  
Cdna Synthesis ◽  
High Quality ◽  
Lemon Balm

<p>Ribonucleic acid (RNA) quality and integrity are crucial for many studies in plant molecular biology. High-quality RNA extraction from plants with high levels of compounds such as polysaccharides, polyphenols, and other secondary metabolites are problematic. RNA extraction from Lemon balm tissues can be difficult due to the presence of polyphenolic and polysaccharide compounds or can be done by expensive protocols. This study shows improvement of a CTAB-based protocol which allows rapid and easy isolation of high-quality RNA from Lemon balm plant. The RNA obtained is suitable for cDNA synthesis and RT-PCR experiments.</p>

scholarly journals High-quality total RNA isolation from melon (Cucumis melo L.) fruits rich in polysaccharides

2017 ◽  
Vol 38 (4) ◽  
pp. 2201 ◽  
Author(s):  
Gabrielle Silveira de Campos ◽  
Ricardo Antônio Ayub ◽  
Rafael Mazer Etto ◽  
Carolina Weigert Galvão ◽  
Marília Aparecida Stroka ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Sodium Perchlorate ◽  
Rna Isolation ◽  
World Market ◽  
Molecular Techniques ◽  
Guanidine Thiocyanate ◽  
High Quality ◽  
Total Rna ◽  
High Concentrations ◽  
Cucumis Melo L

Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazil’s main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1) guanidine thiocyanate/phenol/chloroform; T2) sodium azide/?-mercaptoethanol; T3) phenol/guanidine thiocyanate; T4) CTAB/PVP/?-mercaptoethanol; T5) SDS/sodium perchlorate/PVP/?-mercaptoethanol, and T6) sarkosyl/PVP/guanidine thiocyanate, using the AxyPrepTM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/?-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data.

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 An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene

PLoS ONE ◽  
2021 ◽  
Vol 16 (11) ◽  
pp. e0260002
Author(s):  
María José Cárdenas Espinosa ◽  
Tabea Schmidgall ◽  
Georg Wagner ◽  
Uwe Kappelmeyer ◽  
Stephan Schreiber ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Bacterial Degradation ◽  
Rna Seq ◽  
High Quality ◽  
Total Rna ◽  
Aromatic Substrates ◽  
Next Generation Sequencing Ngs ◽  
Total Rna Extraction

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

scholarly journals A simple and rapid method for isolating high-quality RNA from kenaf containing high polysaccharide and polyphenol contents

2020 ◽  
Author(s):  
Xiaofang Liao ◽  
Hongwei Li ◽  
Aziz Khan ◽  
Yanhong Zhao ◽  
Wenhuan Hou ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Cost Effective ◽  
Spectrophotometric Analysis ◽  
Rt Pcr ◽  
High Quality ◽  
Time Saving ◽  
Ctab Method ◽  
Commercial Kits

AbstractThe isolation of high-quality RNA from kenaf is crucial for genetic and molecular biology studies. However, high levels of polysaccharide and polyphenol compounds in kenaf tissues could irreversibly bind to and coprecipitate with RNA, which complicates RNA extraction. In the present study, we proposed a simplified, time-saving and low-cost extraction method for isolating high quantities of high-quality RNA from several different kenaf tissues. RNA quality was measured for yield and purity, and the proposed protocol yielded high quantities of RNA (10.1-12.9 μg/g·FW). Spectrophotometric analysis showed that A260/280 ratios of RNA samples were in the range of 2.11 to 2.13, and A260/230 ratios were in the range of 2.04-2.24, indicating that the RNA samples were free of polyphenols, polysaccharides, and protein contaminants after isolation. The method of RNA extraction presented here was superior to the conventional CTAB method in terms of RNA isolation efficiency and was more sample-adaptable and cost-effective than commercial kits. Furthermore, to confirm downstream amenability, the high-quality RNA obtained from this method was successfully used for RT-PCR, real-time RT-PCR and Northern blot analysis. We provide an efficient and low-cost method for extracting high quantities of high-quality RNA from plants that are rich in polyphenols and polysaccharides, and this method was also validated for the isolation of high-quality RNA from other plants.

scholarly journals Extraction and purification of RNA from human carious dentine: an approach to enable bacterial gene expression studies

2019 ◽  
Vol 7 (2) ◽  
pp. 145 ◽  
Author(s):  
Daniela Da Silva Bezerra ◽  
Beatriz Gonçalves Neves ◽  
Sarah Florindo de Figueiredo Guedes ◽  
Wanessa Fernandes Matias Regis ◽  
Rafael Nobrega Stipp ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Rna Isolation ◽  
Transcriptional Analysis ◽  
Purification Method ◽  
Bacterial Gene ◽  
Rna Integrity ◽  
Extraction And Purification ◽  
Spin Column ◽  
Dnase Treatment ◽  
Carious Dentine

Background: RNA isolation from bacteria within dentine caries lesions could be difficult due to reduced amount of collectable biomass and high mRNA instability. Attempting to overcome this challenge we describe one protocol developed to extract and purify total RNA from dentine lesions. Objective: customize a bacterial RNA extraction and purification method from human carious dentine. Methods: quantity and purity of extracted RNA were measured with a microvolume UV-VIS spectrophotometer, RNA integrity was assessed by standard denaturing agarose gel electrophoresis and images were captured under ultraviolet light with camera and analyzed. DNase treatment removed genomic DNA and an additional step of purification was carried out in silica spin column.  Results: final yield (ng/μl) was 67.01 ± 22.33, absorbance ratio A260/A280 = 2.0 ± 0.07 and RNA integrity were obtained. The purified samples were reversely transcribed and the expression of atpD and fabM gene from Streptococcus mutans analyzed by quantitative real-time PCR. Conclusion: the extraction methodology developed produced high-quality RNA from dentine microbiota for transcriptional analysis.

scholarly journals A one-tube method for rapid and reliable plant genomic DNA isolation for PCR analysis

2020 ◽  
Author(s):  
Wei Hu ◽  
J. Clark Lagarias
Keyword(s):  
Dna Extraction ◽  
Genomic Dna ◽  
Extraction Method ◽  
Low Cost ◽  
Plant Molecular Biology ◽  
Pcr Analysis ◽  
High Quality ◽  
Plant Dna ◽  
Genomic Dna Isolation ◽  
Dna Extraction Method

AbstractBackgroundConsistent isolation of high quality plant genomic DNA is a prerequisite for successful PCR analysis. Time consumption, ease of operation and procedure cost are important secondary considerations for selecting an effective DNA extraction method. The simple, reliable and rapid DNA extraction method developed by Edwards and colleagues in 1991 [1] has proven to be the gold standard.ResultsThrough modification of the Edwards method of extraction, we have developed a one-tube protocol that greatly improves the efficiency of plant DNA extraction and reduces the potential for sample contamination while simultaneously yielding high quality DNA suitable for PCR analysis. We further show that DNA extracts prepared with this method are stable at room temperature for at least three months.ConclusionThe one-tube extraction method yields high quality plant DNA with improved efficiency while greatly minimizing the potential for cross contamination. This low-cost and environment-friendly method is widely applicable for plant molecular biology research.

scholarly journals Isolation of High Quality RNA for High Throughput Applications From Secondary Metabolite Rich Crocus Sativus L

2021 ◽  
Author(s):  
Zubair Ahmad Wani ◽  
Umer Majeed Wani ◽  
Aabid M Koul ◽  
Asif Amin ◽  
Basit Amin Shah ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Rna Extraction ◽  
Mature Mirnas ◽  
Poor Quality ◽  
Crocus Sativus ◽  
High Quality ◽  
Rna Integrity ◽  
Rna Integrity Number ◽  
Extraction Buffer

Abstract Isolating high quality RNA is a basic requirement while performing high throughput sequencing, microarray and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction that include CTAB, TriZol, SDS based methods, they invariably yield less and poor quality RNA. In the present study we made certain changes in the available protocols including modifications in the extraction buffer and procedure viz-a-viz solutions used for precipitation of RNA. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) greater than 7.5. The quality of the RNA was further assessed by qPCR based amplification of mature miRNAs such as Cs-MIR166c and Cs- MIR396a. In conclusion, the study describes an efficient method of RNA extraction that is highly ideal for high throughput sequencing of small RNAs.

scholarly journals A Simple and Swift Method for Isolating High Quality RNA from Jute (Corchorus spp.)

2011 ◽  
Vol 21 (2) ◽  
pp. 207-211 ◽  
Author(s):  
Niaz Mahmood ◽  
Razib Ahmed ◽  
Muhammad Shafiul Azam ◽  
Haseena Khan
Keyword(s):  
Rna Extraction ◽  
Rna Isolation ◽  
Plant Genome ◽  
Rt Pcr ◽  
High Quality ◽  
Phenol Extraction ◽  
Simultaneous Processing ◽  
Expression Pattern Analysis ◽  
Commercial Kits ◽  
Downstream Processes

High quality RNA extraction is a prerequisite for various types of genetic analyses. Many a time, the existing RNA isolation methods and commercial kits are either time consuming or fail to isolate high quality RNA from plants rich in polysaccharides, oil and other secondary metabolites since different plants contain different amounts of nucleic acids (Khan et al. 2004, Loomis 1974). This problem is particularly acute in case of jute (Corchorus spp.), which is rich in mucilage and other polysaccharides that tends to interfere with the downstream processes (Kundu et al. 2011, Pandey et al. 1996). Several guanidium salt based methods have been successful for RNA isolation from jute seedlings (Khan et al. 2004), but are often cumbersome and expensive; hence limit simultaneous processing of large number of samples. Here we report a simplified and swift protocol for isolating high quality RNA from jute by making key modifications in tissue denaturation and precipitation steps in the protocol described by Ghawana et al. (2011). The protocol allows consistent production of high quality RNA from different species, which makes it particularly suitable for comparative plant genome research. The extraction time has been reduced from two days (for standard guanidium-acid-phenol extraction protocols) to about one hour and the extracted RNA was suitable for downstream processes like cDNA synthesis and expression pattern analysis.   Key words: Jute, Corchorus spp., Swift method, RNA isolation, RT-PCR   D. O. I. 10.3329/ptcb.v21i2.10244   Plant Tissue Cult. & Biotech. 21(2): 207-211, 2011 (December) - Short communication

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