Step 2: RNA extraction and RT-qPCR v1

2021 ◽  
Author(s):  
Peter H L Krijger ◽  
Tim A Hoek ◽  
Sanne Boersma ◽  
Lieke I P M Donders ◽  
Maaike M C Broeders ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Storage System ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Robotic Arms ◽  
Liquid Handling ◽  
2D Barcode ◽  
Simultaneous Processing ◽  
Multiple Sample ◽  
Liquid Handling Robot

STRIP is a start-to-end streamlined and automated procedure for COVID-19 testing, centering on a single Tecan Fluent liquid-handling robot that can process over 14,000 samples per day. Key features of the customized Tecan Fluent robot are (1) on-board 1D and 2D barcode scanners, (2) an automated tube decapper, (3) two robotic arms for simultaneous processing of different procedural steps, (4) a newly-designed spin vessel to keep magnetic beads in solution and immediately transferable to 384-well plates, (5) a built-in magnetic deck and a 384-channel pipetting head for rapid RNA isolation, (6) a heating device for fast drying of RNA prior to elution, (7) a built-in plate sealer and (8) a plate storage system to allow processing of multiple sample plates in a single run (See Materials). Here we describe the RNA extraction and RT-qPCR protocol as currently applied in STRIP.

scholarly journals Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0246302
Author(s):  
Fernando Lázaro-Perona ◽  
Carlos Rodriguez-Antolín ◽  
Marina Alguacil-Guillén ◽  
Almudena Gutiérrez-Arroyo ◽  
Jesús Mingorance ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Low Cost ◽  
Rna Extraction ◽  
Positive Control ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Handling System ◽  
Liquid Handling ◽  
Liquid Handling Robot ◽  
Commercial Kit

Background Two automatable in-house protocols for high-troughput RNA extraction from nasopharyngeal swabs for SARS-CoV-2 detection have been evaluated. Methods One hundred forty one SARS-CoV-2 positive samples were collected during a period of 10-days. In-house protocols were based on extraction with magnetic beads and designed to be used with either the Opentrons OT-2 (OT-2in-house) liquid handling robot or the MagMAXTM Express-96 system (MMin-house). Both protocols were tested in parallel with a commercial kit that uses the MagMAXTM system (MMkit). Nucleic acid extraction efficiencies were calculated from a SARS-CoV-2 DNA positive control. Results No significant differences were found between both in-house protocols and the commercial kit in their performance to detect positive samples. The MMkit was the most efficient although the MMin-house presented, in average, lower Cts than the other two. In-house protocols allowed to save between 350€ and 400€ for every 96 extracted samples compared to the commercial kit. Conclusion The protocols described harness the use of easily available reagents and an open-source liquid handling system and are suitable for SARS-CoV-2 detection in high throughput facilities.

scholarly journals Comparison of Different Kits for SARS-CoV-2 RNA Extraction Marketed in Brazil

2020 ◽  
Author(s):  
Ana Karolina Antunes Eisen ◽  
Meriane Demoliner ◽  
Juliana Schons Gularte ◽  
Alana Witt Hansen ◽  
Karoline Schallenberger ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Rna Extraction ◽  
Severe Pneumonia ◽  
Rna Isolation ◽  
Isolation And Purification ◽  
Silica Column ◽  
Negative Results ◽  
Mean Delay ◽  
Spanish Flu ◽  
The One

ABSTRACTDecember 2019 marked the begining of the greatest pandemic since Spanish Flu, the disease named Covid-19 that cause severe pneumonia. Until May 19, 2020 more than 4 million and 700 thousand cases were oficially notified with about 316 thousand deaths. Etiological agent of the disease was identified as being a new coronavirus, Severe acute respiratory syndrome-related coronavirus (SARS-CoV-2). In this study we compared four different manual methods for RNA isolation and purification for detection of SARS-CoV-2 through qRT-PCR, as well as the extraction quality itself through detection of RNAse P. Magnetic beads-based (MagMax™) and silica column-based (Biopur®) methods presented the better performances. Concerning to the mean delay in CT values when compared to MagMax™, TRIzol™, Biopur® and EasyExtract presented 0,39, 0,95 and 5,23 respectively. Agreement between positive and negative results of different methods when compared with the one with better performance MagMax™ was 94,44% for silica column-based method (Biopur®), 88,89% for phenol-chroloform-based method (TRIzol™) and 77,78% for EasyExtract. We aimed to evaluate how reliable each method is for diagnostic purposes and to propose alternatives when usual methods are not available. In this regard, magnectic beads and silica column-based methods are convenient and reliable choices and phenol-chloroform-based method could also be chosen as an alternative.

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

Bead preparation protocol v1

2021 ◽  
Author(s):  
Peter H L Krijger ◽  
Tim A Hoek ◽  
Sanne Boersma ◽  
Lieke I P M Donders ◽  
Maaike M C Broeders ◽  
...  
Keyword(s):  
Magnetic Particles ◽  
Magnetic Beads ◽  
Liquid Handling ◽  
Liquid Handling Robot

STRIP is a start-to-end streamlined and automated procedure for COVID-19 testing, centering on a single Tecan Fluent liquid-handling robot that can process over 14,000 samples per day. Here we describe the protocol to prepare the magnetic beads that are currently used in STRIP. Note, in this protocol we use Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic) from Cytivia, but other beads such as SeraSil Mag 400 beads (Cytiva) can be used as substitutes.

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 SARS-CoV-2 Direct Detection Without RNA Isolation With Loop-Mediated Isothermal Amplification (LAMP) and CRISPR-Cas12

2021 ◽  
Vol 8 ◽  
Author(s):  
Alfredo Garcia-Venzor ◽  
Bertha Rueda-Zarazua ◽  
Eduardo Marquez-Garcia ◽  
Vilma Maldonado ◽  
Angelica Moncada-Morales ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Direct Detection ◽  
Direct Method ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/μL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.

scholarly journals PlasmoTron: an open-source platform for automated culture of malaria parasites

2017 ◽  
Author(s):  
Theo Sanderson ◽  
Julian C. Rayner
Keyword(s):  
Open Source ◽  
Genetic Modification ◽  
Malaria Parasite ◽  
Scale Up ◽  
Malaria Parasites ◽  
Parasite Culture ◽  
Liquid Handling ◽  
Liquid Handling Robot

We have created a system which allows an inexpensive opensource liquid-handling robot to automate most aspects of bloodstage malaria parasite culture. Parasites are cultured in multiwell microplates, with their details recorded in a database. Information in the database is used to generate commands for the robot to feed, monitor and passage parasite cultures. We show that the system is capable of raising cultures after transfection and then maintaining them at desired parasitaemias, facilitiating significant scale up of both routine culture and experimental genetic modification. The PlasmoTron software is available at plasmotron.org.

scholarly journals Direct-RT-qPCR Detection of SARS-CoV-2 without RNA Extraction as Part of a COVID-19 Testing Strategy: From Sample to Result in One Hour

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 605 ◽  
Author(s):  
Eva Kriegova ◽  
Regina Fillerova ◽  
Petr Kvapil
Keyword(s):  
High Throughput Screening ◽  
High Speed ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Rna Extraction ◽  
Rna Isolation ◽  
High Sensitivity ◽  
Ease Of Use ◽  
Diagnostic Tools ◽  
Heat Inactivation

Due to the lack of protective immunity in the general population and the absence of effective antivirals and vaccines, the Coronavirus disease 2019 (COVID-19) pandemic continues in some countries, with local epicentres emerging in others. Due to the great demand for effective COVID-19 testing programmes to control the spread of the disease, we have suggested such a testing programme that includes a rapid RT-qPCR approach without RNA extraction. The Direct-One-Step-RT-qPCR (DIOS-RT-qPCR) assay detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in less than one hour while maintaining the high sensitivity and specificity required of diagnostic tools. This optimised protocol allows for the direct use of swab transfer media (14 μL) without the need for RNA extraction, achieving comparable sensitivity to the standard method that requires the time-consuming and costly step of RNA isolation. The limit of detection for DIOS-RT-qPCR was lower than seven copies/reaction, which translates to 550 virus copies/mL of swab. The speed, ease of use and low price of this assay make it suitable for high-throughput screening programmes. The use of fast enzymes allows RT-qPCR to be performed under standard laboratory conditions within one hour, making it a potential point-of-care solution on high-speed cycling instruments. This protocol also implements the heat inactivation of SARS-CoV-2 (75 °C for 10 min), which renders samples non-infectious, enabling testing in BSL-2 facilities. Moreover, we discuss the critical steps involved in developing tests for the rapid detection of COVID-19. Implementing rapid, easy, cost-effective methods can help control the worldwide spread of the COVID-19 infection.

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