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 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

scholarly journals An optimized method to obtain high-quality RNA from different tissues in Lilium davidii var. unicolor

2021 ◽  
Author(s):  
Chunlei Wang ◽  
Xuemei Hou ◽  
Nana Qi ◽  
Changxia Li ◽  
Yanyan Luo ◽  
...  
Keyword(s):  
Low Cost ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Extraction Methods ◽  
High Yield ◽  
Rna Seq ◽  
High Quality ◽  
Ctab Method ◽  
Different Tissues ◽  
Lilium Davidii

Abstract Background: The high quality, high yield and purity RNA samples are essential for subsequent molecular experiments such as RT-PCR, qPCR and RNA-seq. However, harvest high quality RNA samples from different tissues in Lilium davidii var. unicolor is a great challenge due to its polysaccharides, polyphenols and other secondary metabolites. In this study, three RNA extraction methods, namely modified TRIzol method, Kit and CTAB methods were reported to obtain the total RNA from Lilium davidii var. unicolor, and the efficient RNA extraction protocol (modified TRIzol method) was described. Results: A Nano drop spectrophotometer and 1% gel electrophoresis were used to detect the RNA quality and integrity. Compared with Kit and CTAB methods, the higher RNA concentrations from different tissues were obtained and the A260/280 ratios of RNA samples were ranged from1.97 to 2.27 when using the modified TRIzol method. None of intact RNA bands were gained from the modified CTAB method, indicating that the RNA samples isolation were degraded. As a result of the modified TRIzol method, the RNA samples in the different tissues from Lilium presented clear 28S and 18S bands.Conclusions: Here, modified TRIzol method is an easy, efficient, and low-cost method for RNA isolation from Lilium davidii var. unicolor. Thus, modified TRIzol method is sufficient to gain eligible RNA to support further molecular experiments in Lilium davidii var. unicolor.

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 A two-pronged approach for rapid and high-throughput SARS-CoV-2 nucleic acid testing

2020 ◽  
Author(s):  
Neeru Gandotra ◽  
Irina Tikhonova ◽  
Nagarjuna R. Cheemarla ◽  
James Knight ◽  
Ellen Foxman ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Low Cost ◽  
Rna Extraction ◽  
Clinical Specimen ◽  
Rna Isolation ◽  
Turnaround Time ◽  
Ease Of Use ◽  
Diagnostic Tools ◽  
Rt Pcr

AbstractImproved molecular screening and diagnostic tools are needed to substantially increase SARS-CoV-2 testing capacity and throughput while reducing the time to receive test results. Here we developed multiplex reverse transcriptase polymerase chain reaction (m-RT-PCR) for detection of SARS-CoV-2 using rapid DNA electrophoresis and alternatively using multiplex viral sequencing (mVseq). For RNA specimens extracted from nasopharyngeal (NP) swabs in viral transport media (VTM), our assays achieved a sensitivity for SARS-CoV-2 detection corresponding to cycle threshold (Ct) of 37.2 based on testing of these specimens using quantitative reverse transcription PCR (RT-qPCR). For NP swab-VTM specimens without prior RNA extraction, sensitivity was reduced to Ct of 31.6, which was due to lower concentration of SARS-CoV-2 genome copies in VTM compared to RNA-extracted samples. Assay turnaround time was 60 minutes using rapid gel electrophoresis, 90 minutes using Agilent Bioanalyzer, and 24-48 hours using Illumina sequencing, the latter of which required a second PCR to produce a sequence-ready library using m-RT-PCR products as the template. Our assays can be employed for high-throughput sequencing-based detection of SARS-CoV-2 directly from a clinical specimen without RNA isolation, while ease-of-use and low cost of the electrophoresis-based readout enables screening, particularly in resource-constrained settings.

scholarly journals Development of new total RNA isolation method for tissues with rich phenolic compounds

2020 ◽  
Vol 45 (4) ◽  
pp. 343-350
Author(s):  
Zafer Seçgin ◽  
Gökhan Gökdemir ◽  
Elif Seda Atabay ◽  
Aslıhan Kurt Kızıldoğan ◽  
Musa Kavas
Keyword(s):  
Phenolic Compounds ◽  
Rna Sequencing ◽  
Rna Isolation ◽  
Wall Structure ◽  
Isolation Method ◽  
High Quality ◽  
Ctab Method ◽  
Total Rna Isolation

AbstractBackgroundRNAs to be used in transcriptome analysis must be of high quality and pure in order to ensure maximum representation of the expressed genes. RNA isolation is difficult in hazelnut tissues containing large amounts of secondary metabolite, phenolic compounds and the cell wall structure. Commonly used protocols for RNA isolation are those that require a lot of labor and time and also do not allow sufficient RNA isolation when applied to tissues rich in phenolic compounds. This study was aimed to develop an efficient method for isolation of total RNAs from bud of hazelnut to be used in RNA sequencing.Materials and methodsAn optimized new method was successfully applied on three different hazelnuts genotypes (Çakıldak, Palaz, Tombul) and about 25 times higher amount of total RNAs per mg fresh tissues were obtained compared to classical CTAB method. Different methods have been tried for the isolation of RNA from hazelnut tissues and the determination of the quality of the obtained RNAs.ResultsThe quality and quantity of isolalated total RNAs were determined by spectrophotometer, electrophoresis and PCR. This success has been caught without any compromise of purity since A260/A280 ratios ranged from 1.90 to 2.04 and A260/A230 ratios were >2.0 in all purified RNAs.ConclusionThe total RNAs isolated with new protocol was found to be suitable for RNA sequencing and other molecular applications.

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.

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 Indirect Laryngeal Surgery in the Clinical Voice Laboratory: The Renewal of a Lost Art

2000 ◽  
Vol 79 (5) ◽  
pp. 350-361 ◽  
Author(s):  
Norman D. Hogikyan ◽  
Melissa Pynnonen
Keyword(s):  
Cost Effectiveness ◽  
General Anesthesia ◽  
Recent Literature ◽  
Low Cost ◽  
Cost Effective ◽  
Topical Anesthesia ◽  
High Quality ◽  
Advantages And Disadvantages ◽  
Laryngeal Surgery ◽  
Type Of Surgery

Since the advent of precision instruments and safe techniques for direct laryngoscopic surgery under general anesthesia, indirect laryngeal surgery has become very uncommon. A review of the recent literature finds that few authors advocate indirect surgery under topical anesthesia, and many otolaryngologists dismiss this technique as being either of only historical interest or an idiosyncratic method practiced only by a handful of clinicians. The societal mandate for cost-effective healthcare and the availability of relatively low-cost, high-quality endoscopes and video equipment warrant a renewed and broader interest in this type of surgery. In this article, we review a series of 27 indirect surgical procedures performed under topical anesthesia in the clinical voice laboratory. We discuss the indications, outcomes, advantages, and disadvantages of this surgery, and we present a brief analysis of its cost-effectiveness. We conclude that indirect laryngeal surgery in the clinical voice laboratory is an effective, safe, efficient, and less costly alternative to some procedures routinely performed under general anesthesia.

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.

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