scholarly journals A Simple and Rapid System for DNA and RNA Isolation from Diverse Plants Using Handmade Kit

2019 ◽  
Author(s):  
Farhad Masoomi-Aladizgeh ◽  
Leila Jabbari ◽  
Reza Khayam Nekouei ◽  
Ali Aalami
Keyword(s):  
Nucleic Acid ◽  
Extraction Method ◽  
Ethylenediaminetetraacetic Acid ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Plant Tissues ◽  
Hexadecyltrimethylammonium Bromide ◽  
Dna And Rna ◽  
Extraction Buffer ◽  
Main Components

Abstract This protocol describes a rapid DNA and RNA extraction method for plant tissues. Hexadecyltrimethylammonium bromide (CTAB), sodium chloride (NaCl), tris base, and ethylenediaminetetraacetic acid (EDTA) are the main components of the extraction buffer. In contrast to all previously reported protocols, this extraction method does not require any stock solutions. This isolation buffer is potential of extracting both DNA and RNA simultaneously. Depending on the purpose of the project, the corresponding steps can be slightly altered to obtain either DNA or RNA. The big advantage of this method is to use general laboratory chemicals to make a powerful extraction buffer, resulting in high quality and quantity nucleic acid. Also, CTAB in this buffer is capable of isolating nucleic acid from recalcitrant plants enriched in secondary metabolites. Importantly, this method is recommended for the projects at which isolating nucleic acid in a short time is of crucial importance. This method probably is usable for all plant tissues and takes about an hour.

scholarly journals Isolation and Detection of Enterovirus RNA from Large-Volume Water Samples by Using the NucliSens miniMAG System and Real-Time Nucleic Acid Sequence-Based Amplification

2005 ◽  
Vol 71 (7) ◽  
pp. 3734-3740 ◽  
Author(s):  
Saskia A. Rutjes ◽  
Ronald Italiaander ◽  
Harold H. J. L. van den Berg ◽  
Willemijn J. Lodder ◽  
Ana Maria de Roda Husman
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Large Volume ◽  
Water Samples ◽  
Extraction Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Rt Pcr ◽  
Virus Rna ◽  
Nasba Assay

ABSTRACT Concentration of water samples is a prerequisite for the detection of the low virus levels that are present in water and may present a public health hazard. The aim of this study was to develop a rapid, standardized molecular method for the detection of enteroviruses in large-volume surface water samples, using a concentration method suitable for the detection of infectious viruses as well as virus RNA. Concentration of water was achieved by a conventional filter adsorption-elution method and ultrafiltration, resulting in a 10,000-fold concentration of the sample. Isolation of virus RNA by a silica-based RNA extraction method was compared with the nonmagnetic and magnetic NucliSens RNA isolation methods. By using the silica-based RNA extraction method in two out of five samples, enterovirus RNA was detected, whereas four out of five samples were positive following RNA isolation with magnetic silica beads. Moreover, estimated RNA levels increased at least 100 to 500 times. Furthermore, we compared enterovirus detection by an in-house reverse transcription (RT)-PCR with a novel commercially available real-time nucleic acid sequence-based amplification (NASBA) assay. We found that the rapid real-time NASBA assay was slightly less sensitive than our in-house RT-PCR. The advantages, however, of a commercial real-time NASBA assay, like the presence of an internal control RNA, standardization, and enormous decrease in turnaround time, makes it an attractive alternative to RT-PCR.

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.

scholarly journals QRREM method for the isolation of high-quality RNA from the complex matrices of coconut

2019 ◽  
Vol 39 (1) ◽  
Author(s):  
Amjad Iqbal ◽  
Yaodong Yang ◽  
Rashad Qadri ◽  
Yi Wu ◽  
Jing Li ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Extraction Method ◽  
Rna Extraction ◽  
The Other ◽  
Plant Tissues ◽  
Betel Nut ◽  
Robust Method ◽  
Plant Metabolites ◽  
High Quality ◽  
Spring Onion

Abstract Complex plant tissues vary in hardness, i.e. some are succulent, while others are complex to break. Besides, plant metabolites, such as polysaccharides, proteins, polyphenols and lipids, can greatly interfere with the RNA extraction. So, in order to obtain a high-quality RNA from the complex tissues (like coconut endosperm, coconut apple and coconut leaf bud) rich in secondary metabolites, a robust method is demanded. Several methods (MRIP, CTAB and TRIZOL) have been used previously for the isolation of quality RNA from the coconut tissues, but without any success. The present study will provide with the details of a new method (Quick and Reliable RNA Extraction Method or QRREM), which have efficiently isolated the intact RNA form the complex tissues of coconut compared with CTAB, Trizol and RNA plant. The method has been validated for the isolation of high-quality intact RNA from the other available plant species (Areca/betel nut, mint and spring onion). The method has various advantages over the other methods in terms of time and cost effectiveness. Furthermore, the resulted RNA from various tissues of coconut performed well in the downstream experiments, i.e. reverse transcription and PCR for the production and amplification of cDNA.

scholarly journals MAVRICS: A Robust and Safe Magnetic Nanoparticle based RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples v2

2021 ◽  
Author(s):  
Mo Li ◽  
Gerardo Ramos-Mandujano
Keyword(s):  
Extraction Method ◽  
Magnetic Nanoparticle ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Emerging Pathogens ◽  
Nucleic Acid Isolation ◽  
Guanidinium Thiocyanate ◽  
Biology Laboratory ◽  
Alternative Solutions ◽  
Basic Equipment

Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities.

scholarly journals Epitachophoresis is a novel versatile total nucleic acid extraction method

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Vladimira Datinska ◽  
Pantea Gheibi ◽  
Keynttisha Jefferson ◽  
Jaeyoung Yang ◽  
Sri Paladugu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Nucleic Acid ◽  
Extraction Method ◽  
Acid Extraction ◽  
Total Nucleic Acid ◽  
Next Generation ◽  
Nucleic Acid Extraction ◽  
Dna And Rna ◽  
Generation Sequencing

AbstractEpitachophoresis is a novel next generation extraction system capable of isolating DNA and RNA simultaneously from clinically relevant samples. Here we build on the versatility of Epitachophoresis by extracting diverse nucleic acids ranging in lengths (20 nt–290 Kbp). The quality of extracted miRNA, mRNA and gDNA was assessed by downstream Next-Generation Sequencing.

scholarly journals Simplified Magnetic Bead Based Viral RNA Extraction for Rapid Detection of COVID-19

2021 ◽  
Author(s):  
Shashi Sharma ◽  
Deepak Pardasani ◽  
Pooja Yadav ◽  
Jyoti S Kumar ◽  
Suman Dhankher ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Extraction Method ◽  
Molecular Detection ◽  
Virus Disease ◽  
Detection System ◽  
Rna Extraction ◽  
Magnetic Bead ◽  
Viral Rna ◽  
Molecular Diagnostic ◽  
Magnetic Extraction

Abstract Rapid and large-scale diagnosis has helped in mitigation the recent ongoing pandemic of corona virus disease of 2019 (COVID-19). The pandemic had a devastating effect on global economy. The molecular detection system has evolved over last two decades and is rapidly replacing the conventional confirmatory techniques in diagnostic virology. However the major limitation in implementation of available molecular detection assays is the non availability of field deployable nucleic acid isolation platform. The standard laboratory diagnosis rely on confirmation of presence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in respiratory specimens of suspected patients. Preparation of viral nucleic acid is a critical step involved followed by downstream molecular diagnostic platforms. For good quality of viral RNA extraction many commercial extraction kits, are available. These are developed in a surge of pandemic scenario keeping in view the large demand for testing. The commercial RNA extraction kits available on either column based or magnetic extraction are limited and, alternative, non-commercial protocols are rapidly required. Here, we have standardized an in-house magnetic bead RNA extraction method which utilises simple in-house reagents and manual extraction method that doesn’t require any high-end equipments. The in-house assay was evaluated against the commercial available silica column and magnetic extraction kits using a panel of 100 throat /nasal swab samples. A high correlation in viral RNA detection with TaqMan qRT-PCR was observed with excellent sensitivity and specificity. Interestingly, the developed method is very simple, cost effective, rapid and can be quickly add up any downstream amplification platform for SARS-CoV-2 detection.

Optimization of the Separation Method for Bioleaching Bacteria DNA and RNA Extracted Simultaneously

2013 ◽  
Vol 825 ◽  
pp. 182-185
Author(s):  
Hui Yun ◽  
Jian Ping Xie ◽  
Ning Huang ◽  
Xin Jie Yang ◽  
Jing Xu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Acid Concentration ◽  
Room Temperature ◽  
Mine Drainage ◽  
Rna Extraction ◽  
Extraction Methods ◽  
Gold Mine ◽  
Dna And Rna ◽  
Nucleic Acid Concentration ◽  
Optimized Conditions

DNA and RNA based analysis was a useful way to characterize microbial communities during biohydrometallurgical processes. However, feasible, affordable and efficient DNA and RNA separation methods are rarely reported although several simultaneous DNA and RNA extraction methods have been developed recently. In this study, various salts including NaCl, CaCl2 and LiCl were tested for separation of DNA and RNA. Salt concentration, nucleic acid concentration, precipitation temperature and time were optimized. The results showed that LiCl was more efficient to separate DNA and RNA than the other two salts. The optimized conditions were as follows: 1/4 volume saturated LiCl was used for precipitating RNA first at -20°C for 30 min for total nucleic acid concentration of approximately 200-400 ng/μL, and then centrifuged at 12,000×g at room temperature for 20 min to collect RNA. DNA in the supernatant was precipitated using 0.6 volume isopropanol and then collected by centrifugation at 12,000×g at room temperature for 20 min. The results indicated that DNA and RNA could be extracted from not only pure culture of Acidithiobacillus ferrooxidans(A.ferrooxidans), Acidithiobacillus caldus(A. caldus), Acidithiobacillus albertensis(A. albertensis), Leptospirillum ferrooxidans(L. ferrooxidans), Ferroplasma thermophilium(F. thermophilium), but also the acid mine drainage(AMD) water samples from Daye copper mine, Xiangxi gold mine, Axi gold mine. The resulted DNA and RNA could be amplified and A260/280 was 1.8-2.0, A260/230 was 1.8-2.2, which indicated high quality of DNA and RNA. This method could be widely used for separation of bioleaching bacteria DNA and RNA extracted simultaneously.

High-resolution nucleic acid sequence mapping via in situ hybridization at the Electron Microscope level

1995 ◽  
Vol 53 ◽  
pp. 752-753
Author(s):  
B.A. Hamkalo ◽  
S. Narayanswami ◽  
A.P. Kausch
Keyword(s):  
Nucleic Acid ◽  
Interphase Nucleus ◽  
Genome Mapping ◽  
Precise Location ◽  
Electron Microscope Level ◽  
Rna Sequences ◽  
Fundamental Interest ◽  
Whole Cells ◽  
Dna And Rna

The availability of nonradioactive methods to label nucleic acids an the resultant rapid and greater sensitivity of detection has catapulted the technique of in situ hybridization to become the method of choice to locate of specific DNA and RNA sequences on chromosomes and in whole cells in cytological preparations in many areas of biology. It is being applied to problems of fundamental interest to basic cell and molecular biologists such as the organization of the interphase nucleus in the context of putative functional domains; it is making major contributions to genome mapping efforts; and it is being applied to the analysis of clinical specimens. Although fluorescence detection of nucleic acid hybrids is routinely used, certain questions require greater resolution. For example, very closely linked sequences may not be separable using fluorescence; the precise location of sequences with respect to chromosome structures may be below the resolution of light microscopy(LM); and the relative positions of sequences on very small chromosomes may not be feasible.

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