A robust plant RNA isolation method suitable for Affymetrix GeneChip analysis and quantitative real-time RT-PCR

2009 ◽  
Vol 4 (3) ◽  
pp. 333-340 ◽  
Author(s):  
Damla D Bilgin ◽  
Evan H DeLucia ◽  
Steven J Clough
Keyword(s):  
Real Time ◽  
Rna Isolation ◽  
Affymetrix Genechip ◽  
Rt Pcr ◽  
Isolation Method ◽  
Genechip Analysis

Total RNA‐Isolation of Abdominal Hernia of Rats for Quantitative Real‐Time Reverse Transcription (RT) PCR Assays

2007 ◽  
Vol 38 (1) ◽  
pp. 87-93
Author(s):  
Christian Morsczeck ◽  
Michael Korenkov ◽  
Manfred Nagelschmidt ◽  
Domonkos Feher ◽  
Jörg Michael Schierholz
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Rna Isolation ◽  
Abdominal Hernia ◽  
Rt Pcr ◽  
Total Rna ◽  
Pcr Assays ◽  
Total Rna Isolation

scholarly journals Modified protocol for RNA isolation from different parts of field-grown jute plant suitable for NGS data generation and quantitative real-time RT-PCR

2019 ◽  
Vol 18 (27) ◽  
pp. 647-658 ◽  
Author(s):  
Ahmed Rasel ◽  
Sabbir Hossain Md. ◽  
Samiul Haque Md. ◽  
Monjurul Alam Md. ◽  
Shahidul Islam Md.
Keyword(s):  
Real Time ◽  
Rna Isolation ◽  
Data Generation ◽  
Rt Pcr ◽  
Ngs Data ◽  
Different Parts

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.

Total RNA-Isolation of Abdominal Hernia of Rats for Quantitative Real-Time Reverse Transcription (RT) PCR Assays

2008 ◽  
Vol 38 (2) ◽  
pp. 129-135
Author(s):  
Christian Morsczeck ◽  
Michael Korenkov ◽  
Manfred Nagelschmidt ◽  
Domos Feher ◽  
Jörg Michael Schierholz
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Rna Isolation ◽  
Abdominal Hernia ◽  
Rt Pcr ◽  
Total Rna ◽  
Pcr Assays ◽  
Total Rna Isolation

scholarly journals Automation of Nucleic Acid Isolation on KingFisher Magnetic Particle Processors

2007 ◽  
Vol 12 (4) ◽  
pp. 195-201 ◽  
Author(s):  
Xingwang Fang ◽  
Roy C. Willis ◽  
Angela Burrell ◽  
Kurt Evans ◽  
Quoc Hoang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Pathogen Detection ◽  
Magnetic Particle ◽  
Rna Isolation ◽  
Viral Rna ◽  
Cell Fractionation ◽  
Rt Pcr ◽  
Total Rna ◽  
Total Rna Isolation

We describe automated nucleic acid (NA) isolation from diverse sample types using MagMAX kits (Ambion, Inc.) on KingFisher Magnetic Particle Processors (Thermo Scientific). The MagMAX-96 Blood RNA Isolation Kit is designed for total RNA isolation from whole blood from several species, without white blood cell fractionation, in about 45 min (including genomic DNA removal). The MagMAX-96 Total RNA Isolation Kit is designed for total RNA isolation from up to 2 × 10 6 cultured cells and up to 10-mg tissue. The isolated total RNA is highly intact and pure, ready to use in downstream applications such as microarray analysis or real-time reverse transcription (RT)-PCR for gene expression profiling or pathogen detection. The MagMAX-96 Viral RNA Isolation Kit is designed for viral RNA and DNA isolation from cell free or nearly cell-free samples such as swabs, serum, and plasma; it takes about 15 min. Total NA of high quality and purity is recovered at >75% efficiency, providing high sensitivity for pathogen detection by real-time RT-PCR. Unlike automated liquid handling systems that move reagents into and out of a single well of a multiwell plate to perform the different steps of an RNA isolation procedure, the KingFisher Magnetic Particle Processors use permanent magnetic rods to collect magnetic beads from solution and release them into another well containing reagent for the subsequent step of the procedure. The effectiveness of bead collection and transfer lead to superior washing and elution efficiency, as well as rapid processing. It is a very effective strategy for automation of magnetic-bead-based NA isolation kits. (JALA 2007; 12:195–201)

scholarly journals Selection of RNA isolation method for molecular detection of grapevine viruses

2017 ◽  
Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽  
pp. 267-270
Author(s):  
P. Komínek
Keyword(s):  
Extraction Method ◽  
Molecular Detection ◽  
Rna Isolation ◽  
Rt Pcr ◽  
Isolation Method ◽  
Phloem Tissue ◽  
Total Rna ◽  
Chloroform Extraction ◽  
Selection Of

Grapevines infected with Grapevine leafroll-associated virus-1 (GLRaV-1) and Grapevine leafroll-associated virus-3 (GLRaV-3) were selected. Total RNA was isolated from grapevine phloem tissue scrapped from dormant canes by three different methods: extraction with urea buffer followed with phenol-chloroform extraction, method using Concert<sup>TM</sup> reagent (Invitrogen) followed with chloroform-isopropylalcohol extraction, and procedure using RNeasy Plant Mini Kit (Qiagen). The highest yield of RNA was obtained using Concert<sup>TM</sup> reagent. If this RNA was used in RT-PCR, GLRaV-1 and GLRaV-3 were easily detected. From RNA isolated by other two methods these viruses were not detected.

scholarly journals Assessment of the use and quick preparation of saliva for rapid microbiological diagnosis of COVID-19

2020 ◽  
Author(s):  
Mariana Fernández-Pittol ◽  
Juan Carlos Hurtado ◽  
Estela Moreno-García ◽  
Elisa Rubio ◽  
Mireia Navarro ◽  
...  
Keyword(s):  
Real Time ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Heat Inactivation ◽  
Rt Pcr ◽  
Elution Volume ◽  
Heated Sample ◽  
Predictive Values ◽  
One Step ◽  
Sensitivity Specificity

AbstractThe objective of this study was to assess the performance of direct real time RT-PCR detection of SARS-CoV-2 in heated saliva samples, avoiding the RNA isolation step. Oropharyngeal and nasopharyngeal swabs together with saliva samples were obtained from 51 patients clinically diagnosed as potentially having COVID-19. Two different methods were compared: 1. RNA was extracted from 500 μl of sample using a MagNA Pure Compact Instrument with an elution volume of 50μl and 2. 700µL of saliva were heat-inactivated at 96°C for 15 minutes, and directly subjected to RT-PCR. One step real time RT-PCR was performed using 5 μl of extracted RNA or directly from 5 μl of heated sample. RT-PCR was performed targeting the SARS-CoV-2 envelope (E) gene region. Diagnostic performance was assessed using the results of the RT-PCR from nasopharyngeal and oropharyngeal swabs as the gold standard. The overall sensitivity, specificity, positive and negative predictive values were 81.08%, 92.86%, 96.77% and 65.00%, respectively when RNA extraction was included in the protocol with saliva, whereas sensitivity, specificity, positive and negative predictive values were 83.78%, 92.86%, 68.42% and 96.88%, respectively, for the heat-inactivation protocol. However, when the analysis was performed exclusively on saliva samples with a limited time from the onset of symptoms (<9 days, N=28), these values were 90%, 87.5%, 44% and 98.75% for the heat-inactivation protocol. The study showed that RT-PCR can be performed using saliva in an RNA extraction free protocol, showing good sensitivity and specificity.

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