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 Isolation of functional total RNA from Tilia cordata leaves and pollen

2012 ◽  
Vol 77 (8) ◽  
pp. 1003-1012
Author(s):  
Jana Ognjenovic ◽  
Omar Tantoush ◽  
Ratko Jankov ◽  
Cirkovic Velickovic ◽  
Jelena Vukmirica
Keyword(s):  
Molecular Biology ◽  
Genomic Dna ◽  
Tilia Cordata ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
High Quality ◽  
Total Rna ◽  
Dna Contamination ◽  
Successful Isolation ◽  
Synthesis Reactions

Conditions for isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing Qiagen plant mini kit while total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIZOL? preparation of total RNA. Total RNA isolated using TRIZOL? was contaminated with genomic DNA but treatment with enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, conditions for elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. Isolated total RNA from both leaves and pollen was used successfully in firstand second-strand cDNA synthesis reactions, as well as, in RT-PCR, demonstrating that the total RNA isolated using this method is functional. In conclusion, pure and functional total RNA from Tilia cordata leaves and pollen (27.8 ? 7.9?g/g leaves; 25.7 ? 1.1?g/g pollen) can be obtained and applicable for further molecular biology studies.

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 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 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 NaCl-Induced Elicitation Alters Physiology and Increases Accumulation of Phenolic Compounds in Melissa officinalis L.

2021 ◽  
Vol 22 (13) ◽  
pp. 6844
Author(s):  
Barbara Hawrylak-Nowak ◽  
Sławomir Dresler ◽  
Maria Stasińska-Jakubas ◽  
Magdalena Wójciak ◽  
Ireneusz Sowa ◽  
...  
Keyword(s):  
Salt Stress ◽  
Phenolic Compounds ◽  
Secondary Metabolites ◽  
Defense Mechanism ◽  
Chemical Diversity ◽  
Melissa Officinalis ◽  
Herbal Products ◽  
Ps Ii ◽  
Lemon Balm ◽  
Abiotic Elicitor

In nature, plants usually produce secondary metabolites as a defense mechanism against environmental stresses. Different stresses determine the chemical diversity of plant-specialized metabolism products. In this study, we applied an abiotic elicitor, i.e., NaCl, to enhance the biosynthesis and accumulation of phenolic secondary metabolites in Melissa officinalis L. Plants were subjected to salt stress treatment by application of NaCl solutions (0, 50, or 100 mM) to the pots. Generally, the NaCl treatments were found to inhibit the growth of plants, simultaneously enhancing the accumulation of phenolic compounds (total phenolics, soluble flavonols, anthocyanins, phenolic acids), especially at 100 mM NaCl. However, the salt stress did not disturb the accumulation of photosynthetic pigments and proper functioning of the PS II photosystem. Therefore, the proposed method of elicitation represents a convenient alternative to cell suspension or hydroponic techniques as it is easier and cheaper with simple application in lemon balm pot cultivation. The improvement of lemon balm quality by NaCl elicitation can potentially increase the level of health-promoting phytochemicals and the bioactivity of low-processed herbal products.

scholarly journals IDENTIFIKASI TOMATO INFECTIOUS CHLOROSIS VIRUS PENYEBAB PENYAKIT KLOROSIS PADA TANAMAN TOMAT DI CIPANAS JAWA BARAT MELALUI PERUNUTAN NUKLEOTIDA GEN PROTEIN SELUBUNG UTAMA

2015 ◽  
Vol 15 (1) ◽  
pp. 33
Author(s):  
Fitrianingrum Kurniawati ◽  
Gede Suastika ◽  
Giyanto .
Keyword(s):  
Coat Protein ◽  
Coat Protein Gene ◽  
Protein Gene ◽  
Rna Extraction ◽  
Dna Amplification ◽  
Causal Agent ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Polymerase Chain ◽  
Tomato Infectious Chlorosis Virus

Identification of tomato infectious chlorosis virus, the causal agent of chlorosis disease on tomato in Cipanas West Java by sequencing of main coat protein gene nucleotide. Tomato infectious chlorosis virus (TICV) causes chlorosis on tomato. Tomatoes infected by this virus shows interveinal yellowing, necrotic, bronzing, brittleness, and declining in productivity. This study aims to identify the causal agent of chlorotic disease on tomato by sequencing the coat protein gene. The methods involve collecting infected plants, total RNA extraction, cDNA synthesis, DNA amplification, visualization of the results of reverse transcription polymerase chain reaction (PCR), and phylogenetic analysis using BLAST, clustal w, Bioedit v 7.0.5.3, MEGA v 6:06. RT-PCR using spesific primers (CP-F TICV Bam and TICV R-Hind) amplified a DNA band of 792 bp, which has been successfully sequenced and identified as TICV. Nucleotide sequences homology analysis showed that TICV Indonesia_TWJ isolate Cipanas is the same strain as TICV from other countries (99.4 – 100%), such as Spain, Greece, USA, France, and Italy.

scholarly journals Isolation of high-quality RNA from plant seeds

2021 ◽  
Vol 66 (2) ◽  
Author(s):  
Alisa Mishko ◽  
Maria Sundyreva ◽  
Ilya Stepanov ◽  
Sergey Efimenko ◽  
Vladimir Plotnikov ◽  
...  
Keyword(s):  
Plant Material ◽  
Rna Extraction ◽  
Fruit Tree ◽  
Future Research ◽  
Minimum Level ◽  
Rt Pcr ◽  
High Quality ◽  
Simple Method ◽  
Plant Seeds ◽  
Extraction Buffer

The apple (Malus domestica Borkh.) is one of the major fruit tree crops, but it hasn’t been well-studied as a breeding object for molecular investigations. It is important to develop reliable and rapid methods that allow the preparation of plant material for future research. We introduce a quick and simple method for isolating high-quality RNA from lipid-rich apple seeds (M. domestica cv. Golden Delicious). Our method does not employ highly toxic reagents, because we exclude phenol, 2-mercaptoethanol and others. The chemical composition of the extraction buffer is simple and has a minimum level of toxicity. We showed that, in chaotropic conditions (i.e., with lithium chloride-urea), silica (SiO2) can bind with the lipids and RNA will remain in the solution. The extracted RNA was of high quality and we successfully used it for synthesizing cDNA and RT-PCR. The protocol developed by us can be useful for researchers working with RNA extraction from plant seeds.

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