To determine virus titers from 50 mg of lung tissue, tissue homogenates were prepared using a bead mill (Analytic Jena) and 10-fold serial dilutions were prepared in MEM, which were then added to Vero E6 cells in 12-well plates. The dilutions were removed after 2 hours and cells were overlaid with 1.25% microcrystalline cellulose (Avicel) in MEM supplemented with 10% FBS and penicillin/streptomycin. Two days later, cells were formalin fixed, stained with crystal violet, and plaques were counted. RNA was extracted from 25 mg of lung homogenates and oropharyngeal swabs using the innuPREP Virus RNA kit (Analytic Jena). Viral RNA copies were quantified in 10% of the obtained eluate volume with a 1-step RT-qPCR reaction using a standard curve and the Luna Universal Probe One-Step RT-qPCR kit (New England Biolabs) and previously published TaqMan primers and probe [69 (link)] on a StepOnePlus RealTime PCR System (Thermo Fisher Scientific).
Innuprep virus rna kit
Manufactured by Analytik Jena
Sourced in Germany
The InnuPREP Virus RNA kit is a laboratory equipment designed for the extraction and purification of viral RNA from various sample types. The kit utilizes a silica-based membrane technology to efficiently capture and purify viral RNA, which can then be used for downstream applications such as RT-qPCR or reverse transcription.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Neb luna universal probe one step rt qpcr kit, by New England Biolabs (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Bead mill, by Analytik Jena (2 mentions)
Rq1 rnase free dnase, by Promega (2 mentions)
Rneasy plus mini kit, by Qiagen (2 mentions)
Luna universal probe one step rt qpcr kit, by New England Biolabs (1 mentions)
Avicel, by FMC Biopolymer (1 mentions)
Nanophotometer p class p 300, by Implen (1 mentions)
8 protocols using innuprep virus rna kit
1
SARS-CoV-2 Viral Titer and RNA Quantification
2
RVA Genotyping from Preserved Stool
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Preserved stool samples were transported to the Institute of Hygiene and Tropical Medicine in Lisbon, Portugal for RVA genotyping. Viral RNA was extracted from 10% (w/v) stool suspensions using the innuPREP Virus RNA Kit (Analytik Jena AG, Jena, Germany), according to the manufacturer’s instructions. The RNA was eluted in RNase-free water (60 μl) and stored at -80°C until further use.
3
SARS-CoV-2 Virus Titer Quantification
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To determine virus titers from 50 mg of lung tissue, tissue homogenates were prepared using a bead mill (Analytik Jena, Jena, Germany), and 10-fold serial dilutions were prepared in MEM, which were then added to Vero E6 cells in 12-well plates. The dilutions were removed after 2 h and cells were overlaid with 1.25% microcrystalline cellulose (Avicel, FMC BioPolymer, Hamburg, Germany) in MEM supplemented with 10% FBS and penicillin/streptomycin. Three days later, cells were formalin-fixed, stained with crystal violet and plaques were counted. RNA was extracted from 25 mg of lung homogenates and oropharyngeal swabs using the innuPREP Virus RNA kit (Analytik Jena, Jena, Germany). Viral RNA copies were quantified in 10% of the obtained eluate volume with a one-step RT-qPCR reaction using a standard curve and the NEB Luna Universal Probe One-Step RT-qPCR kit (New England Biolabs, Ipswich, MA, USA) and previously published TaqMan primers and probe (SARS-CoV-2 E_Sarbeco) [20 (link)] on a StepOnePlus RealTime PCR System (Thermo Fisher Scientific, Waltham, MA, USA).
4
SARS-CoV-2 RNA Quantification in Lung and Swabs
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RNA was extracted from 25 mg lung homogenates and oropharyngeal swabs using the innuPREP Virus RNA kit (Analytik Jena AG). SARS-CoV-2 RNA copies were quantified using the NEB Luna Universal Probe One-Step RT-qPCR kit (New England Biolabs) on a StepOnePlus RealTime PCR System (Thermo Fisher Scientific) as previously described (Corman et al., 2020 ).
5
Quantifying vTR and hTR Expression in MDV-Infected Cells
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vTR and hTR expression levels were quantified in MDV-infected cells and tumor tissues using RT-qPCR. Briefly, 106 CECs were infected with 103 PFU of wt, v∆vTR, or vhTR. Total RNA was isolated from infected CECs and tumor tissues using the RNeasy Plus Mini Kit (Qiagen) and the innuPREP Virus RNA kit (Analytik Jena GmbH), respectively, according to the manufacturer’s instructions. The isolated RNAs were treated with RQ1 RNase-Free DNase (Promega), and reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). vTR and hTR expression was quantified using TaqMan qPCR, and the cellular GAPDH reference gene was used to normalize vTR and hTR expression. The vTR, hTR, and GADPH primer and probe sets have been previously published (14 (link), 21 (link)) and are shown in Table 1 .
6
Quantifying vTR and hTR Expression in MDV-Infected Cells
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vTR and hTR expression levels were quantified in MDV-infected cells and tumor tissues using RT-qPCR. Briefly, 106 CECs were infected with 103 PFU of wt, v∆vTR, or vhTR. Total RNA was isolated from infected CECs and tumor tissues using the RNeasy Plus Mini Kit (Qiagen) and the innuPREP Virus RNA kit (Analytik Jena GmbH), respectively, according to the manufacturer’s instructions. The isolated RNAs were treated with RQ1 RNase-Free DNase (Promega), and reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). vTR and hTR expression was quantified using TaqMan qPCR, and the cellular GAPDH reference gene was used to normalize vTR and hTR expression. The vTR, hTR, and GADPH primer and probe sets have been previously published (14 (link), 21 (link)) and are shown in Table 1 .
7
SARS-CoV-2 RNA Quantification from Lungs
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RNA was extracted from 25 mg of lung homogenates and tracheal swabs using the innuPREP Virus RNA Kit (Analytik Jena). SARS-CoV-2 RNA copies were quantified using the NEB Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) on the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific) as previously described (42 (link)).
8
RNA Extraction and cDNA Synthesis
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RNA extraction for in vitro experiments was performed on cell lysates or supernatants using the NucleoSpin RNA minikit for RNA purification (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. The concentration of RNA extracted from cell lysates was measured using a P‐class P 300 NanoPhotometer (Implen GmbH, Munich, Germany).
Retrotranscription to cDNA was performed with 500 ng of intracellular RNA or 10 μL of RNA from supernatants, using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions.
For in vivo samples, RNA was extracted from 25 mg of lung homogenates and oropharyngeal swabs using the innuPREP virus RNA kit (Analytik Jena, Jena, Germany) according to the manufacturer’s instructions.
Retrotranscription to cDNA was performed with 500 ng of intracellular RNA or 10 μL of RNA from supernatants, using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions.
For in vivo samples, RNA was extracted from 25 mg of lung homogenates and oropharyngeal swabs using the innuPREP virus RNA kit (Analytik Jena, Jena, Germany) according to the manufacturer’s instructions.
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