VSV-Sars-S-GFP was quantified via qRT-PCR. Virally infected tissues (olfactory mucosa) were harvested and snap frozen. Tissues were homogenized with zirconia/silica beads using a tissue beater in RNA lysis buffer (Qiagen). Homogenates were centrifuged for 10 min at 1500 x g/4°C. Viral RNA was extracted from clarified supernatant using QIAamp Viral RNA kit (see Key Resources Table ). Quantitative PCR was performed using EXPRESS One-Step Superscript qRT-PCR kit (see Key Resources Table ) and using 18S rRNA as an endogenous control (ThermoFisher Scientific, #4319413E) using the QuantStudio 6 pro instrument (ThermoFisher). Gene expression was plotted on an arbitrary standard curve then normalized to 18S in the same sample using QuantStudio Design and Analysis Software (ThermoFisher). Quantification of viral reads (in duplicates) was performed with the following custom primers and probes (IDT) (Sequences provided in the Key Resources Table ) : SARS CoV-2 S FWD, SARS CoV-2 S REV, SARS CoV-2 S Probe, Eukaryotic 18s rRNA Endogenous Control
Quantstudio 6 pro instrument
Manufactured by Thermo Fisher Scientific
The QuantStudio 6 Pro instrument is a real-time PCR system designed for reliable, accurate, and sensitive nucleic acid analysis. It features a modular design, flexible block formats, and advanced thermal cycling capabilities to support a variety of applications, including gene expression analysis, SNP genotyping, and pathogen detection.
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Lab products found in correlation
Quantstudio design and analysis software, by Thermo Fisher Scientific (1 mentions)
Express one step superscript qrt pcr kit, by Thermo Fisher Scientific (1 mentions)
Rna lysis buffer, by Qiagen (1 mentions)
5 mm stainless steel bead, by Qiagen (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Powertrack sybr green master mix kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using quantstudio 6 pro instrument
1
Quantitative Analysis of SARS-CoV-2 Spike Expression
2
Kidney Gene Expression Analysis
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Using realtime PCR, transcription of genes, Ccl2, c-Myc, Col1a2, Ctgf, Socs3, Stat3, and Tgf-b, was determined in relation to a house keeping gene, Gapdh, and expression levels calculated using 2-ΔΔCt values. Kidney tissue was homogenized using 5mm stainless steel beads (#69989) and TissueLyser LT (#85600), all from Qiagen, Helden, Germany. Total RNA was extracted from the homogenates using RNeasy Kit (#74106, Qiagen), following manufacturer’s instructions. RNA, 500ng per each tissue homogenate, was transcribed into cDNA using QuantiTect Reverse Transcription kit (#205311, Qiagen). Gene expression was determined in triplicates using PowerTrack SYBR Green Master Mix kit (#A46113, Thermo Fisher Scientific) and the QuantStudio 6 Pro instrument (#A43161, Thermo Fisher Scientific) for realtime PCR. Data were analyzed using the QuantStudio design and analysis software version 2.5.0. Primer sequences are shown in the key resources table , primer specificity was ensured by determining DNA melting curves.
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