Qiaamp viral rna kit

Manufactured by Qiagen

Sourced in Germany, United States, France, Canada, United Kingdom, Spain, Switzerland, China

The QIAamp Viral RNA kit is a product designed for the rapid and efficient purification of viral RNA from various sample types, including cell-free body fluids, tissue samples, and cultured cells. The kit utilizes a spin-column format and a specialized buffer system to ensure high-quality RNA extraction, making it suitable for a range of downstream applications, such as RT-PCR and RT-qPCR analysis.

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Close spelling variants of this product

QIAamp kit (104 citations) QIAamp Viral RNA (30 citations) QIAamp RNA kit (12 citations) QIAamp Viral kit (4 citations) QIAamp Viral RNA Mini KitTM (2 citations)

543 protocols using qiaamp viral rna kit

RNA Extraction from Cell Culture and Clinical Samples

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Supernatants from cultured MDCK cells were centrifuged at 10,000 ×
g for 10 min. Viral RNA was prepared from 140 μL of the supernatant using a QIAamp^® Viral RNA Kit (QIAGEN) according to the manufacturer's instructions with the slight modification that the viral RNA was eluted in 70 μL AVE buffer (QIAGEN).
Total RNA was prepared from the clinical specimens using a QIAamp^® Viral RNA Kit (QIAGEN) (using 140 μL of clinical specimen) or MagMAX™ 96 Viral Isolation Kit (Thermo Fisher Scientific) (using 50 μL of clinical specimen) with KingFisher Flex (Thermo Fisher Scientific) according to the manufacturers’ instructions. Total RNA from the 140-μL clinical specimens was eluted with 60 μL AVE buffer (QIAGEN), whereas total RNA from the 50-μL clinical specimens was eluted with 30 μL elution buffer (Thermo Fisher Scientific).

Nakauchi M., Takayama I., Takahashi H., Oba K., Kubo H., Kaida A., Tashiro M, & Kageyama T. (2014). Real-time RT-PCR assays for discriminating influenza B virus Yamagata and Victoria lineages. Journal of Virological Methods, 205, 110-115.

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Viral RNA Extraction from Sewage Samples

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Viral RNA was extracted from each sewage sample after centrifugation, to produce a solid fraction, and after PEG precipitation of the supernatant, to produce a PEG-precipitated fraction, following the procedures of previous studies with minor modifications (Jones and Johns, 2009 (link); Kitamura et al., 2021 (link)). Specifically, 160 ml of each sample was divided equally into four aliquots (40 ml each) held in 50 ml tubes and centrifuged at 10,000 × g for 30 min. RNA was extracted from the resulting pellet (solid fraction sample) using the NucleoBond RNA Soil kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions. Meanwhile, the entire supernatant was precipitated using PEG 8000 (final concentration 10%; Promega, Madison, WI, United States) and NaCl (final concentration 1 M; Wako, Tokyo, Japan) by incubating at 4°C overnight with gentle rotation. After centrifugation at 10,000 × g for 60 min, the precipitate was resuspended in 500 μl of phosphate buffer solution (pH 7.0, 0.067 mol/L; Nacalai Tesque, Kyoto, Japan). RNA was extracted from 140 μl of the PEG-precipitated suspension using a QIAamp Viral RNA Kit (Qiagen, Hilden, Germany). RNA was also extracted from 140 μl of raw unconcentrated sewage samples using a QIAamp Viral RNA Kit (Qiagen).

Tanimoto Y., Ito E., Miyamoto S., Mori A., Nomoto R., Nakanishi N., Oka N., Morimoto T, & Iwamoto T. (2022). SARS-CoV-2 RNA in Wastewater Was Highly Correlated With the Number of COVID-19 Cases During the Fourth and Fifth Pandemic Wave in Kobe City, Japan. Frontiers in Microbiology, 13, 892447.

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Extraction and Analysis of COVID-19 RNA

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RNA from inactivated swab nasopharyngeal samples were obtained from COVID-19 patients and donated by Hospital Ramón y Cajal (Madrid). RNA was extracted using QIAamp Viral RNA Qiagen kit and the total RNA obtained was eluted in water free of RNase. Its concentration was measured using a Nanodrop, then it is storage at – 80 °C. To avoid any cross-contamination between samples and/or during their manipulation by the operator, all the procedure were performed in P2-biosecurity cabinets with spatial and temporal separation between COVID-19 positive and negative samples. Specifically, the positive sample was analyzed by RT-qPCR with a Ct value of 25. Negative sample has a Ct value above the threshold (∼35).
The samples were obtained with the consent of all participants and approved by “Comité de Ética de la Investigación con Medicamentos del Hospital Universitario Ramón y Cajal”. Reference: 127–21.

Kaci K., del Caño R., Luna M., Milán-Rois P., Castellanos M., Abreu M., Cantón R., Galán J.C., Somoza Á., Miranda R., González de Rivera G., García-Mendiola T, & Lorenzo E. (2022). Paving the way to point of care (POC) devices for SARS-CoV-2 detection. Talanta, 247, 123542.

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COVID-19 Patient RNA Extraction and RT-PCR

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RNAs of 192 clinical samples with positive results from RT-PCR tests were extracted using the QIAamp Viral RNA Qiagen kit, from nasopharyngeal samples provided by Imam Reza Hospital (Mashhad, Iran) from unnamed COVID-19 patients. The prepared samples were kept in a freezer at −80 °C before use. A commercial one-step Taqman PCR Time Real PCR, Pishtaz Teb Diagnostic COVID-19 RT-PCR Kit was then used in the RT-PCR tests (Pishtaz Teb, Iran). The nasopharyngeal, saliva, and serum samples of healthy people were obtained from Imam Reza Hospital and stored in RNase-free microtubes at 4 °C until use. The positive nasopharyngeal samples were then inactivated by heating them at 56 °C for 30 min, and all real samples were diluted 20 times with PBS (pH 7, 0.05 M).

Bolourinezhad M., Rezayi M., Meshkat Z., Soleimanpour S., Mojarrad M., zibadi F., Aghaee-Bakhtiari S.H, & Taghdisi S.M. (2023). Design of a rapid electrochemical biosensor based on MXene/Pt/C nanocomposite and DNA/RNA hybridization for the detection of COVID-19. Talanta, 265, 124804.

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COVID-19 Nasopharyngeal Sample RNA Extraction

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RNA from inactivated swab nasopharyngeal samples obtained from COVID-19 patients’ and donated by Hospital Ramón y Cajal (Madrid) was extracted with the QIAamp Viral RNA Qiagen kit. The total RNA obtained was eluted in water free of RNase, and its concentration was measured using a Nanodrop prior to its storage at – 80 °C. To avoid any cross-contamination between samples and/or during their manipulation by the operator, all the procedure were performed in P2-biosecurity cabinets with spatial and temporal separation between COVID-19 positive and negative samples. Specifically, the positive sample was analysed by RT-qPCR with a Ct value of 13. Negative sample has a Ct value above the threshold (~35).

Pina-Coronado C., Martínez-Sobrino Á., Gutiérrez-Gálvez L., Del Caño R., Martínez-Periñán E., García-Nieto D., Rodríguez-Peña M., Luna M., Milán-Rois P., Castellanos M., Abreu M., Cantón R., Galán J.C., Pineda T., Pariente F., Somoza Á., García-Mendiola T., Miranda R, & Lorenzo E. (2022). Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures for sensitive and selective SARS-CoV-2 sensing. Sensors and Actuators. B, Chemical, 369, 132217.

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RNA Purification and Northern Blotting

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At 9 dpi, total cellular RNA was purified using a GeneJET RNA Purification Kit (Thermo Fisher), according to the manufacturer’s protocol. The purified RNA was analyzed by Northern blotting, as described [16 (link)]. For detection of HDV RNA in the cell culture medium, RNA purification was performed using the QIAamp Viral RNA Kit (Qiagen), according to the manufacturer’s instructions. Viral RNA was then analyzed by Northern blotting. The detection of HBV DNA in 181 cell culture supernatants was carried out as described previously [16 (link)].

Aronthippaitoon Y., Szerman N., Ngo-Giang-Huong N., Laperche S., Ungeheuer M.N., Sureau C., Khamduang W, & Gaudy-Graffin C. (2023). Are International Units of Anti-HBs Antibodies Always Indicative of Hepatitis B Virus Neutralizing Activity?. Vaccines, 11(4), 791.

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RT-qPCR Quantification of CTV RNA

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An RNA standard was constructed by initially reverse transcribing the CTV RNA. Subsequently, a 349 nucleotide-long sequence from the CTV ORF1 helicase region between nucleotides 3217 and 3565 was amplified and cloned into a pDrive vector (Qiagen, Düsseldorf, Germany). Following transformation into XL10 Gold cells, the plasmid containing the specific insert was isolated and linearized, and the region of interest was transcribed using a T7 High Yield RNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA). Prior to quantification, CTV RNA was extracted using the QIA-amp Viral RNA Kit (Qiagen). The extracted samples were prepared for quantification using the iTaq Universal One-Step RT-qPCR Kit (Bio-Rad, Hercules, CA, USA). The primers used amplify the genomic region between nucleotides 3247 and 3409: (Forward) 5′-ggcaaccatcctctacaaacac-3′ and (Reverse) 5′-gatgtcttgtgggagcctgtag-3′. The thermal cycling conditions were 50 °C for 10 min, 95 °C for 1 min, and 40 cycles of 95 °C for 10 s, 65 °C for 20 s, and 72 °C for 40 s.

von Nordheim M., Boinay M., Leisi R., Kempf C, & Ros C. (2016). Cutthroat Trout Virus—Towards a Virus Model to Support Hepatitis E Research. Viruses, 8(10), 289.

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Virus Titration Assay using CTV RNA Quantification

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For virus titration, an infectivity assay based on CTV RNA quantification was developed. Columns 2–11 of two 96 well plates were seeded with 4 × 10⁴ RTGill-W1 cells in Leibovitz’s L-15 media. One week later, serial dilutions of cell culture supernatant containing CTV (4 × 10⁶ genome copies/µL) were added in 8-plicates (50 µL in 2% MEM per well). The starting dilution was 10⁻², with a serial dilution factor of five. One day after the infection, the media was removed, the cells were washed, and 200 µL of fresh 2% MEM was added per well. After two weeks, the plates were frozen to −70 °C to lyse the cells. RNA was extracted from samples (140 µL) by using the QIAamp viral RNA kit (QIAGEN) and quantified as specified above. The infectious titer was determined by the Spearman–Kärber method and expressed as log TCID₅₀/mL.

von Nordheim M., Boinay M., Leisi R., Kempf C, & Ros C. (2016). Cutthroat Trout Virus—Towards a Virus Model to Support Hepatitis E Research. Viruses, 8(10), 289.

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Isolation and Infection of Primary Mouse Myofibroblasts

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Primary MTFs were isolated from the tails of male WT C57BL/6 mice aged 10 weeks, as previously described (Salmon, 2005). Virus infections were performed with the respective CHIKV nsP‐mutants at a multiplicity of infection (MOI) of 10 in serum‐free Dulbecco's modified Eagle medium (DMEM; HyClone). Virus overlay was removed after incubation for 1.5 h at 37°C and replenished with fresh DMEM supplemented with 10% fetal bovine serum (FBS; HyClone). Mock infections were carried out in parallel. Infected MTFs were harvested 12 h post‐infection (hpi) for infectivity quantification. CHIKV infection was directly quantified based on ZsGreen signal detection under the FITC channel (Lum et al, 2018), and data were acquired with a BD FACSCanto II flow cytometer (BD Biosciences). Viral RNA was extracted from 140 μl cell mixture using a QIAamp viral RNA kit (Qiagen). The viral load was determined by quantitative reverse transcription PCR (qRT–PCR) with a probe that detects the nsP1 encoding part of CHIKV genomic RNA (Plaskon et al, 2009).

Chan Y., Teo T., Utt A., Tan J.J., Amrun S.N., Abu Bakar F., Yee W., Becht E., Lee C.Y., Lee B., Rajarethinam R., Newell E., Merits A., Carissimo G., Lum F, & Ng L.F. (2019). Mutating chikungunya virus non‐structural protein produces potent live‐attenuated vaccine candidate. EMBO Molecular Medicine, 11(6), e10092.

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Genome-wide Sequencing of HIV-1 Envelope

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HIV-1 RNA was isolated from plasma using the Qiagen QIAamp viral RNA kit and reverse transcribed to cDNA using SuperScript III reverse transcriptase (Invitrogen, CA). The envelope genes were amplified from single-genome templates (68 (link)), and amplicons were directly sequenced using the ABI PRISM BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA) and resolved on an ABI 3100 automated genetic analyzer. The full-length env sequences were assembled and edited using Sequencher version 4.5 software (Genecodes, Ann Arbor, MI). Multiple sequence alignments were performed using Muscle (69 (link)) and edited with AliView (70 (link)).

Anthony C., York T., Bekker V., Matten D., Selhorst P., Ferreria R.C., Garrett N.J., Karim S.S., Morris L., Wood N.T., Moore P.L, & Williamson C. (2017). Cooperation between Strain-Specific and Broadly Neutralizing Responses Limited Viral Escape and Prolonged the Exposure of the Broadly Neutralizing Epitope. Journal of Virology, 91(18), e00828-17.

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