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 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.

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 Big data from small tissues: extraction of high-quality RNA for different plant tissue types during oilseed Brassica spp. seed development for RNA-Sequencing

2019 ◽  
Author(s):  
Laura Siles ◽  
Peter Eastmond ◽  
Smita Kurup
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
Seed Development ◽  
Developmental Stages ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Extraction Methods ◽  
High Quality ◽  
Gene Expression Analyses ◽  
Rna Extraction Methods

AbstractObtaining high-quality RNA for gene expression analyses from different seed tissues is challenging due to the presence of various contaminants, such as polyphenols, polysaccharides and lipids which interfere with RNA extraction methods. At present, the available protocols for extracting RNA from seeds require high amounts of tissue and are mainly focused on extracting RNA from whole seeds. However, extracting RNA at the tissue level enables more detailed studies regarding tissue specific transcriptome during development. Seeds from heart stage embryo to mature developmental stages of Brassica napus and B. oleracea were sampled for isolation of the embryo, endosperm and seed coat tissues. Ovules and gynoecia wall tissue were also collected at the pre-fertilization stage. After testing several RNA extraction methods, E.Z.N.A. Plant RNA Kit and Picopure RNA Isolation kit extraction methods with some modifications, as well as the use of PVPP for seed coats and endosperms at green stages, resulted in high RNA concentrations with clear 28S and 18S bands and high RIN values. Here, we present efficient and reliable RNA extraction methods for different genotypes of Brassica spp for different tissue types during seed development. The high-quality RNA obtained by using these methodologies is suitable for RNA-Sequencing and gene expression analyses.

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 Impact of RNA Extraction and Target Capture Methods on RNA Sequencing Using Formalin-Fixed, Paraffin Embedded Tissues

2019 ◽  
Author(s):  
Christopher A. Hilker ◽  
Aditya V. Bhagwate ◽  
Jin Sung Jang ◽  
Jeffrey G Meyer ◽  
Asha A. Nair ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Gene ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Rna Seq ◽  
Gene Detection ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Whole Transcriptome ◽  
Formalin Fixed

AbstractFormalin fixed paraffin embedded (FFPE) tissues are commonly used biospecimen for clinical diagnosis. However, RNA degradation is extensive when isolated from FFPE blocks making it challenging for whole transcriptome profiling (RNA-seq). Here, we examined RNA isolation methods, quality metrics, and the performance of RNA-seq using different approaches with RNA isolated from FFPE and fresh frozen (FF) tissues. We evaluated FFPE RNA extraction methods using six different tissues and five different methods. The reproducibility and quality of the prepared libraries from these RNAs were assessed by RNA-seq. We next examined the performance and reproducibility of RNA-seq for gene expression profiling with FFPE and FF samples using targeted (Kinome capture) and whole transcriptome capture based sequencing. Finally, we assessed Agilent SureSelect All-Exon V6+UTR capture and the Illumina TruSeq RNA Access protocols for their ability to detect known gene fusions in FFPE RNA samples. Although the overall yield of RNA varied among extraction methods, gene expression profiles generated by RNA-seq were highly correlated (>90%) when the input RNA was of sufficient quality (≥DV200 30%) and quantity (≥ 100 ng). Using gene capture, we observed a linear relationship between gene expression levels for shared genes that were captured using either All-Exon or Kinome kits. Gene expression correlations between the two capture-based approaches were similar using RNA from FFPE and FF samples. However, TruSeq RNA Access protocol provided significantly higher exon and junction reads when compared to the SureSelect All-Exon capture kit and was more sensitive for fusion gene detection. Our study established pre and post library construction QC parameters that are essential to reproducible RNA-seq profiling using FFPE samples. We show that gene capture based NGS sequencing is an efficient and highly reproducible strategy for gene expression measurements as well as fusion gene detection.

scholarly journals Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10340
Author(s):  
Pacharaporn Angthong ◽  
Tanaporn Uengwetwanit ◽  
Wirulda Pootakham ◽  
Kanchana Sittikankaew ◽  
Chutima Sonthirod ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Genomic Dna ◽  
Extraction Methods ◽  
Marine Organisms ◽  
High Quality ◽  
Sequencing Platform ◽  
Global Food Security ◽  
Long Read ◽  
Ctab Method ◽  
Global Food

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.

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