Water samples were treated with methods described by Maunula et al. (2012) with some modifications. In general, 5 l of the seawater samples were prefiltered through a Waterra ® filter (FHT-700) (Powell et al. 2000) , but in some cases, only 1 or 3 l could pass through the filter. Filtering was continued through a GF/F membrane (Whatman International). Virus particles were eluted from the Waterra filter using 50 ml of 50 mM glycine-3% beef extract (pH 9.5) and from the GF/F membrane with 1 ml AVL lysis buffer (Qiagen) after shaking for 10 min at room temperature. Both eluates were subjected to RNA extraction with a Viral RNA Mini Kit (Qiagen). Aquarium water from the infection trials and the liquid waste samples were not filtered. For determining the presence of VHSV, 140 µl of each sample were collected for RNA extraction with a Qiagen Viral RNA Mini Kit. Sediment samples were diluted by taking 5 g of each sample and adding 1 ml of phosphate-buffered saline. Suspensions were briefly stirred, and 200 µl of the liquid were taken for RNA extraction, which was performed using a Nu clisens magnetic extraction kit (Biomérieux). RNA was analysed using qRT-PCR both undiluted and in 1:10 dilution (RNase-free water).
Viral rna mini kit
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom, Japan, France, Canada
The Viral RNA Mini Kit is a laboratory product designed for the rapid and efficient extraction of viral RNA from various sample types. It utilizes a silica-membrane-based technology to selectively bind and purify viral RNA, which can then be used for downstream applications such as RT-PCR analysis.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (23 mentions)
Cfx96 detection system, by Bio-Rad (13 mentions)
Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (12 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (9 mentions)
One step probe qrt pcr kits, by Qiagen (8 mentions)
Lightcycler 96 system, by Roche (7 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (7 mentions)
Onestep rt pcr kit, by Qiagen (6 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
Rneasy kit, by Qiagen (5 mentions)
Sars cov 2 rna, by Twist Bioscience (5 mentions)
Qiacube, by Qiagen (5 mentions)
Avl lysis buffer, by Qiagen (4 mentions)
Taqman rna to ct 1 step kit, by Thermo Fisher Scientific (4 mentions)
Random primers, by Evrogen (4 mentions)
Nuclease free water, by Evrogen (4 mentions)
Dna ladder, by Evrogen (4 mentions)
Mmlv reverse transcription kit, by Evrogen (4 mentions)
Onestepplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Tamra, by Thermo Fisher Scientific (4 mentions)
268 protocols using viral rna mini kit
1
Viral RNA Extraction from Aquatic Samples
2
Viral RNA Extraction from Rectal Swabs
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Viral RNA extraction from rectal swabs was performed using a slightly modified version of the Qiagen Viral RNA Mini Kit protocol (Qiagen GmbH, Hilden, Germany). Firstly, 200 μL of each sample in 800 μL of AVL lysis buffer (Qiagen GmbH, Hilden, Germany) was heat inactivated at 70 °C for 10 min. After pulse vortexing and short centrifugation, samples in pools of seven comprising 100 μL of each sample were extracted by the subsequent steps outlined in the Qiagen Viral RNA Mini Kit protocol and eluted in a final volume of 100 μL.
3
DENV Detection in Mosquitoes and Larvae
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RNA was extracted from individual mosquito’s head-thorax region and from larvae pool (10 larvae/pool) using the Qiagen Viral RNA Mini Kit (Qiagen, cat. # 52906), according to the manufacturer’s instructions. DENV infection in the extracted RNA of individual mosquitoes, as well as larval pool was assessed by one-step reverse transcription-PCR (Thermo Fisher Scientific, Cat. # 12594025) targeting the capsid-pre-membrane (C-prM) gene region with primers and methods as described (Lanciotti et al., 1992 (link)). The 511-bp PCR product of the DENV-positive individuals was gel purified, sequenced in both directions, and used to identify and characterize circulating serotypes and genotypes.
4
Multiplex Viral Pathogen Detection Protocol
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RNA was purified using the Viral RNA Mini Kit (Qiagen). Testing was carried out using identical laboratory methods in both study sites by real-time RT-PCR using the Qiagen One-Step RT-PCR System and previously published assays for influenza A and B viruses (Flu A/B) [10] (link), respiratory syncytial virus (RSV) [11] (link), human metapneumovirus (hMPV) [12] (link), human parainfluenza viruses 1–4 (hPiV1–4) [13] (link), [14] (link), human coronaviruses (hCoVs) -229E, -OC43, -NL63 and -HKU1 [15] (link), enteroviruses (EV) [16] (link), rhinoviruses (RhV) [17] (link) and adenoviruses (AdV) [18] (link). Assay sensitivity and specificity were evaluated on photometrically quantified in vitro transcribed cRNA/DNA controls generated from cloned PCR amplicons containing the respective genomic target sites as described previously [19] (link). In Ghana, multiplex reactions were used to identify Flu A and Flu B, as well as RSV and hMPV, without the possibility of distinguishing individual viruses due to limited resources. RSV and hMPV are genetically related and constitute the Paramyxoviridae subfamily Pneumovirinae; from hereon they are referred to as pneumoviruses. Enveloped viruses under study included Flu A/B, pneumoviruses, hPiV1–4 and hCoVs. Non-enveloped viruses included EV, RhV and AdV.
5
SARS-CoV-2 Detection in Respiratory Samples
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Following collection, MTNS were preserved in a DNA/RNA Shield medium that inactivates all infectious pathogens. MTNS collected during clinic visits were sent to the North Carolina State Laboratory for Public Health for detection of SARS-CoV-2 virus using the CDC Influenza SARS-CoV-2 (Flu SC2) multiplex polymerase chain reaction (PCR) assay [26 ]. MTNS collected at home by participants between clinic visits were stored at –20°C (253.15 K) upon receipt by study staff. Using a Qiagen Viral RNA Mini Kit [27 ], RNA was extracted from 200μl(mm3) of the DNA/RNA shield, then processed for SARS-CoV-2 PCR testing using the TaqPath™ COVID‑19 Combo Kit [28 ].
6
Optimized Viral Metagenomics Sample Preparation
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An optimized sample preparation protocol for viral metagenomics—NetoVIR [61 (link)] was used to analyze the mosquito pools and individuals as well as a negative control. Briefly, whole mosquitoes were homogenized with 200 μl PBS in a MINILYS tissue homogenizer for 1 min at 3000 rpm using 2.8 nm ceramics beads (Precellys) and centrifuged (17,000 g for 3 min), and 150 μl supernatant were then used for filtration (0.8 μm pore size) to enrich for viral particles. The filtrate was then treated with a cocktail of Benzonase (Novagen) and Micrococcal Nuclease (New England Biolabs) in a homemade buffer (1 M Tris, 100 mM CaCl2, and 30 mM MgCl2) to digest free-floating nucleic acids. DNA and RNA were extracted (QIAGEN Viral RNA mini kit), reverse-transcribed, and randomly amplified using a slightly modified Whole Transcriptome Amplification 2 (WTA2) Kit procedure (Sigma-Aldrich). WTA2 products were purified, and the libraries were prepared for Illumina sequencing using the NexteraXT Library Preparation Kit (Illumina). A cleanup after library synthesis was performed using a 1.8 ratio of Agencourt AMPure XP beads (Beckman Coulter, Inc.). Sequencing of the samples was performed on a NextSeq500 High throughput platform (Illumina) for 300 cycles (2 × 150 bp paired ends) (Additional file 2 ).
7
Quantifying HIV-1 RNA from Crushed ARV Tablets
8
Viral RNA Extraction from Liver
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The RNA was extracted from 140 µl of liver homogenate supernatant using a Qiagen Viral RNA Mini kit, according to the manufacturer’s instructions. Elution was performed in 60 µl.
9
Viral Screening of Serum Samples
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All specimens collected were screened for HBV, HCV and HEV with both serologic and molecular tools. HAV was screened using only molecular methods. Prior to molecular analysis, nucleic acids of all serum samples were extracted and purified with the Viral RNA Mini kit (Qiagen, Hilden, Germany). Extraction was done following the manufacturer’s protocol.
10
ZIKV RNA Quantification from Serum
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