Total RNA was extracted from mouse lung homogenates, and viral RNA was extracted from cleared mouse lung homogenates and cell culture supernatants using the following commercially available kits according to the manufacturer’s instructions: for viral RNA extraction, QIAamp Viral RNA Mini Kit (QIAGEN) and E.Z.N.A Viral RNA kit (Omega Bio-tek) were used. Quantification of viral RNA was performed by quantitative reverse transcription PCR (RT-qPCR) as described below. For total RNA extraction, the samples were passed through a QIAshredder (QIAGEN) to reduce viscosity and subsequently RNA was isolated using the ROTI Prep RNA Mini kit (Carl Roth). After isolation, RNA concentration was measured using a NanoVue Plus Spectrophotometer (GE Healthcare). For cDNA synthesis, 0.5 µg RNA from each sample was reverse-transcribed using the qScript cDNA Synthesis Kit (Quantabio). For subsequent qPCR reactions, PerfeCTa SYBR Green SuperMix (Quantabio) was used in a total reaction volume of 15 µl. TaqMan primer/probes for mouse inflammatory genes were purchased from Microsynth and are listed below. RT-PCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems) with the following conditions: 95°C for 3 min, followed by 45 amplification cycles (15 s at 95°C) and elongation (1 min at 60°C).
E z n a viral rna kit
Manufactured by Omega Bio-Tek
Sourced in United States
The E.Z.N.A Viral RNA kit is a product offered by Omega Bio-Tek for the extraction and purification of viral RNA from various sample types. It utilizes a silica-based membrane technology to effectively isolate viral RNA, making it suitable for downstream applications such as reverse transcription and PCR analysis.
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39 protocols using e z n a viral rna kit
1
Quantitative analysis of viral and host gene expression
2
SARS-CoV-2 Viral Load Quantification
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SARS-CoV-2 RNA viral loads were determined from nasal swabs sampled in 500 µL QVL Lysis Buffer containing 10 µg/mL carrier RNA (Poly A) (Omega Bio-Tek). MS2 Phage Control (TaqMan™ 2019-nCoV Control Kit v2 (Applied Biosystems)) was added to each sample prior to RNA extraction as an internal positive control. RNA isolation was performed using the E.Z.N.A.® Viral RNA Kit (Omega Bio-Tek). This was followed by multiplex qRT-PCR of the isolated RNA on a QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher) using the TaqMan™ 2019-nCoV Control Kit v2 (Applied Biosystems), including serial dilutions of known SARS-CoV-2 viral RNA ranging from 1 × 104 copies/µL to 1 × 100 copies/µL. Viral loads were calculated based on the standard curve and expressed as copies/µL.
3
Plasma-based HTLV-1 tax gene detection
4
Viral RNA Extraction and cDNA Synthesis
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Three-hundred ml of body effusion samples and suspensions of rectal swab samples from FIP-suspected cases were prepared as 10% (w/v) suspensions with sterile phosphate-buffered saline. The fluid was clarified by centrifugation at 1,000 g for 10 min, and the supernatant was collected [27 (link)]. Viral RNA was extracted using a viral RNA purification kit according to the manufacturer’s instructions (E.Z.N.A. Viral RNA Kit, Omega Bio-tek, GA, USA). Five ml of RNA-containing sample was added to the premix of the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific Inc, MA, USA) for reverse transcription (cDNA synthesis) with random hexamers, following the manufacturer’s instructions.
5
Viral Load and CD4+ T Cell Decline
6
Quantifying Zika Virus Infectivity
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The amount of the cell-free infectious virus in the supernatant of infected cells was quantified using a focus-forming unit assay, as previously described [14 ]. Mouse anti-Flavivirus group antigen antibody, clone D1-4G2-4-15 (Millipore) was used as primary antibody and donkey anti-mouse IgG antibody conjugated with horseradish peroxidase (Jackson Research Laboratories) served as the secondary antibody. Extracellular viral RNA was extracted from the supernatant of infected cells or serum from infected animals using an E.Z.N.A. viral RNA kit (Omega Bio-Tek) and used to quantify the viral genome present via the following RT-qPCR protocol. Extracted RNA was reverse transcribed using iScript cDNA synthesis (Bio-Rad). Primers Zika1087 (5′-CCGCTGCCCAACACAAG-3′), Zika1163c (5′-CCACTAACGTTCTTTTGCAGACAT-3′), and FAM-tagged probe Zika1108FAM (5′-AGCCTACCTTGACAAGCAGTCAGACACTCAA-3′) were used in combination with a standard curve spanning 107 copies/reaction to 1 copy/reaction to quantify ZIKV genome copies via qPCR. Transformed data were presented as (log10) viral genome copies per microliter.
7
Ducklings Immunized with rDHAV-1: Viral Dissemination
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Ducks received analgesia (carprofen, 5 mg kg−1, i.p.) before the start of surgery. Ducks were anaesthetized with isoflurane (3.5%) and maintained under anaesthesia (2–2.5%) throughout the inoculation. Fifty 1-day-old healthy ducklings were injected subcutaneously with 0.25 ml of 104.0 LD50 of rDHAV-1. The heart, liver, kidney, spleen, thymus and bursa of Fabricius (BF) of the 1-day-old ducklings were collected at 1, 6, 12, 18, 24, 48 and 72 h post inoculation. According to the manufacturer′s instruction, virus RNA was isolated from tissue samples using the E.Z.N.A.™ Viral RNA Kit (Omega Bio-Tek, Doraville, USA) and then were measured by qRT-PCR. Non-template control (NTC) samples were included in each run.
The animal experiments were carried out in accordance with the guidelines issued by Shandong Agricultural University Animal Care and Use Committee (SDAUA-2014-014).
The animal experiments were carried out in accordance with the guidelines issued by Shandong Agricultural University Animal Care and Use Committee (SDAUA-2014-014).
8
Full-Length Genomic Amplification of Swine Coronaviruses
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According to the instruction manual, viral RNA was extracted using the E.Z.N.A Viral RNA Kit (OMEGA Bio-Tek). The cDNA was synthesized using Reverse Transcriptase M-MLV (Takara, Dalian, China) and Random Primer (Takara, Dalian, China). We designed 12 and 10 pairs of specific primers for full-length genomic amplification of TGEV and PDCoV positive samples, respectively (Tables S1 and S2 ), by Platinum SuperFi II high-fidelity DNA polymerase (Invitrogen, Waltham, MA, USA). PCR amplification products were ligated using DNA A-Tailing Kit (Takara, Dalian, China) and pMD18-T vectors (Takara, Dalian, China), and finally, all plasmids were sent to Tsingke Biotechnology Co., Ltd. (Beijing, China). for Sanger sequencing.
9
Viral RNA Extraction and Segment Amplification
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Viral RNA was extracted from the allantoic supernatant using E. Z. N. A. Viral RNA kit (Omega Bio-Tek) according to the manufacturer guidelines. Viral segments were amplified by RT-PCR using Superscript III one-step RT-PCR kit (Invitrogen) and segment specific primers (supplementary table 5 ). RT-PCR products were gel purified (QIAGEN). pDZ plasmid was digested with SapI restriction enzyme (New England biolabs) and treated with alkaline phosphatase (New England biolabs) and gel purified. Purified RT-PCR products were cloned into digested pDZ plasmid using In-Fusion kit (Takara bio). Plasmid sequences were confirmed by Sanger sequencing (Psomogen).
10
Enhancing EV-A71-B5 Resistance to E151
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E151-sensitive EV-A71-B5 was serially passaged in Vero cells in the presence of 10 μM E151 10 times and then in the presence of 100 μM E151 another 4 times. The fourteenth passaged virus, named Vero/B5-E151-P14, was selected, as it was E151-enhanced in Vero cells. The genomic RNA of EV-A71-B5 and Vero/B5-E151-P14 was extracted using the E.Z.N.A. viral RNA kit (Omega Bio-tek), and the P1 genes were amplified by one-step RT-PCR kit (Qiagen) using the primers seqEV-A71-P1-F and seqEV-A71-P1-R. The RT-PCR thermal cycling conditions were applied at an initial incubation at 50°C for 30 min (reverse transcription), 95°C for 15 min (initial PCR activation step), followed by 40 cycles: 94°C for 30 s (denaturation), 58°C for 30 s (annealing), and 68°C for 4 min (extension), and a final extension at 68°C for 10 min. The PCR products were purified after gel electrophoresis and inserted into pJET1.2 vector (Thermo Scientific) for sequencing. The P1 sequences were compared, and the mutations responsible for E151 enhancement were further confirmed by reverse genetics.
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