To generate neutralization escape mutants, CHIKV 181/25 (105 FFU) was incubated with 10 μg/mL of anti-CHIKV mAbs for 1 h at 37°C. The virus–mAb complexes were added to Vero cells. One day post-infection, the virus supernatant was removed and then incubated with mAbs for 1 h at 37°C and added to new Vero cells. This process was repeated for 5 days. Viral RNA from bulk supernatant was extracted with MagMAX Viral RNA extraction kit using a Kingfisher Flex instrument (Thermo Fisher) according to the manufacturer’s instructions. cDNA was generated using ProtoScript® II First Strand cDNA Synthesis Kit (NEB) according to the manufacturer’s instructions using random hexamers. Viral structural genes were amplified with the following primers: 5′-TGCCATTCCAGTTATGTGCC-3′ and 5′-CACGCATAGCACCACGATTA-3′, purified with ChargeSwitch PCR clean-up kit (Thermo Fisher) and subject to long-read amplicon sequencing, Oxford Nanopore (Plasmidsaurus).
Magmax viral rna extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MagMAX Viral RNA Extraction Kit is a magnetic bead-based assay designed for the rapid and efficient extraction of viral RNA from a variety of sample types. The kit utilizes a simple, automated workflow to isolate high-quality RNA that is suitable for downstream molecular analyses.
Automatically generated - may contain errors
Lab products found in correlation
Kingfisher flex instrument, by Thermo Fisher Scientific (1 mentions)
Chargeswitch pcr clean up kit, by Thermo Fisher Scientific (1 mentions)
Protoscript 2 first strand cdna synthesis kit, by New England Biolabs (1 mentions)
Kingfisher 96 magnetic particle handling system, by Thermo Fisher Scientific (1 mentions)
Magattract 96 cador pathogen kit, by Qiagen (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Rna ultrasense one step quantitative rt pcr system, by Thermo Fisher Scientific (1 mentions)
Dtx 880 multimode detector, by Beckman Coulter (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
4 protocols using magmax viral rna extraction kit
1
Generating CHIKV Neutralization Escape Mutants
2
DNA Extraction and Nucleic Acid Purification
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DNA for the specificity panel was extracted using the Power-Soil DNA isolation kit (MOBIO), as per manufacturer’s instructions, with the following modifications at the elution stage: 30 μl of nuclease free water, pre-heated to 30°C, was added to the white filter membrane, followed by an incubation period of five minutes at room temperature.
Nucleic acid for rtPCR and lab based VDN LAMP was extracted and purified from collected samples using the MagMax Viral RNA extraction kit (Thermo Fisher Scientific), and Kingfisher-96 magnetic particle handling system (Thermo Fisher Scientific), as per manufacturer’s instructions.
Nucleic acid for rtPCR and lab based VDN LAMP was extracted and purified from collected samples using the MagMax Viral RNA extraction kit (Thermo Fisher Scientific), and Kingfisher-96 magnetic particle handling system (Thermo Fisher Scientific), as per manufacturer’s instructions.
3
RNA Extraction from Tissue Samples
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Tissue samples (spleen, cervical, mandibular, mesenteric lymph nodes, and Peyer's patches) collected at necropsy were homogenized in 1 mL MEM per 100 mg of tissue using stainless steel beads in the Tissuelyser II (Qiagen, USA). A 250 µL aliquot of tissue lysate was added to 750 µL of Trizol LS reagent and incubated for 5 min at room temperature. The collected serum was treated similarly. Bromochlorophenol (100 µL) was added to the mixture to separate the aqueous phase. The samples were shaken vigorously for 15 sec and incubated at RT for 5 min. The mixture was centrifuged at 12,000 RCF for 15 min at 4°C and the aqueous layer was transferred to a clean tube. For RNA extraction, 100 µL of the aqueous layer was used for both of the following 2 extraction protocols. A King Fisher automated magnetic particle processor (Thermo Fisher Scientific) and the MagMAX Viral RNA extraction kit (Ambion, USA) in trial 1, and a Qiagen BioSprint 96 automated magnetic particle processor (Thermo Fisher Scientific) and the MagAttract 96 cador pathogen kit (Qiagen) in trial 2 were used according to the manufacturer's recommendations.
4
RVFV Infection Assay with Compounds
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Vero cells were pretreated for 2 hours with equivalent volumes of either DMSO (control), 17-AAG (10 µM), BAPTA (10 µM), or KNK437 (10 µM), prior to infection with the MP-12 strain of RVFV at an MOI of 0.1. Following infection, cells were washed with PBS and media containing either DMSO or compound was added back to the cells. Twenty-four hours later, supernatants were collected for analysis of viral RNA. Viral RNA was extracted using Ambion’s (Applied Biosystems/Ambion, Austin, TX) MagMAX viral RNA extraction kit and quantitated using q-RT-PCR with primers and probe for G2, originally described by Drosten et al[31] (link). Q-RT-PCR assays were performed using Invitrogen’s (Carlsbad, CA) RNA UltraSense One-Step Quantitative RT-PCR System on an ABI 7000 sequence detection system. Absolute quantitation of RVFV RNA was performed using a standard curve where the RNA concentration is expressed in genomic copies/reaction. Currently, our assay detects 102 copies per reaction 100% of the time and 101 copies 50% of the time, which is in agreement with other published methods of PCR detection [32] (link). Cell viability assays were performed using CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. DTX 880 multimode detector (Beckman Coulter) was used for detection of Luminescence.
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