RNA was extracted with the Nucleospin TriPrep (Macherey-Nagel) and the QIASymphony RNA (Qiagen) kits according to the manufacturers’ instructions. Affymetrix HG U133 plus 2.0 arrays were prepared and scanned according to the manufacturer’s protocol and as reported previously.18 (link)Differential gene expression analyses and exploratory gene set enrichment analyses (GSEA) were performed after Robust Multi-array Average (RMA) normalization19 (link) and batch correction, with the Partek Genomics suite platform (v 6.6). Analyses were performed with the Broad Institute MySig libraries of curated gene sets C1–C7 version 5.0,20 (link) 1000 permutations and default additional parameters. An false discovery rate (FDR) threshold of 0.1 was applied.
Qiasymphony rna
Manufactured by Qiagen
Sourced in Germany
The QIAsymphony RNA is a fully automated sample processing system designed to extract and purify RNA from various sample types. It offers a standardized and reproducible RNA extraction workflow, enabling efficient processing of multiple samples simultaneously.
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5 protocols using qiasymphony rna
1
RNA Extraction, Microarray, and Differential Gene Expression Analysis
2
Quantification of Influenza and Immune Markers
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RNA was extracted from THP-1 or A549 cells using automated RNA extraction system QIAsymphony RGQ (Qiagen, Germany) with QIAsymphony RNA extraction kits as per the kit's protocol. To quantify the influenza viral load, proinflammation cytokine (IL-6 and IP-10), apoptosis-associated (TRAIL and FAS) gene, C5a receptor (C5aR1 and C5aR2) and CRP receptor (FCGR2 and FCGR3) mRNA levels, a quantitative real-time RT-PCR was performed on a real-time PCR detection system (Agilent Technologies Inc., Santa Clara, CA). The primer and probe sets were described in our previous report (Gao et al., 2013b (link)) or from commercial products (Hs02375669, Hs01906226, Hs04206243, Hs04211858; Life Science Technologies, USA).
3
Transcriptome Analysis of De Novo Glioblastoma
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Seventy-six fresh-frozen surgical samples of de novo GBMs were prospectively collected between 2010 and 2015. RNA was extracted with the Nucleospin® TriPrep (Macherey-Nagel, Düren, Germany) and the QIASymphony RNA (Qiagen, Venlo, The Netherlands) kits according to the manufacturers’ instructions. Affymetrix HG U133 plus 2.0 arrays were prepared and scanned according to the manufacturer’s protocol and as reported previously [54 (link)]. Quality control and differential gene expression analyses were performed with R (v3.2.2). Based on principal component analysis (PCA) plots, 3 outliers were removed. Robust Multi-array Average (RMA) normalization was applied. Batch correction was performed with the ‘sva’ package. Differential expression was analyzed with the ‘limma’ package and heatmaps were created with the ‘heatmap3′ package (R).
Exploratory Gene Set Enrichment Analyses (GSEA) were performed after RMA-normalization [55 (link)] and batch correction, with the Partek® Genomics suite platform (v6.6) (Partek, St. Louis, MO, USA). Analyses were performed with the Broad Institute MySig libraries of curated gene sets C1—C7 version 5.0 [56 (link)], 1000 permutations and default additional parameters. An FDR threshold of 0.25 was applied as recommended [55 (link)].
Exploratory Gene Set Enrichment Analyses (GSEA) were performed after RMA-normalization [55 (link)] and batch correction, with the Partek® Genomics suite platform (v6.6) (Partek, St. Louis, MO, USA). Analyses were performed with the Broad Institute MySig libraries of curated gene sets C1—C7 version 5.0 [56 (link)], 1000 permutations and default additional parameters. An FDR threshold of 0.25 was applied as recommended [55 (link)].
4
SARS-CoV-2 Detection in Saliva Samples
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Viral RNA extraction from prepared saliva samples was performed according to the manufacturer's instructions using the QIAsymphony RNA (Qiagen, Hilden, Germany) kit. After isolation of viral RNA, amplification of ORF1 ab and N gene fragments of SARS CoV-2 virus was performed with the SARS-CoV-2 Double Gene RT-qPCR (Bio-Speedy-USHAŞ, Ankara, Turkey) kit. Ct-values below 38 were considered positive, and the Ct-values were recorded numerically as a quantitative indicator of viral load (Ct-value/saliva). Viral RNA isolation and amplification were successfully performed in saliva samples of 125 patients, and three patients had Ct-values above 38. The patients with a Ct-value above 38 in saliva were followed up, but were excluded from statistical analyzes regarding clinical course.
5
MERS-CoV Nucleic Acid Extraction and RT-PCR Detection
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Upon receipt, samples were shacked vigorously and equal volumes of sample and external lysis buffer (6 M Guanidine Isothiocyanate, 30% Triton X-100, 100 mM Tris–HCl, 0.01% Bromophenol blue) were mixed in a safety cabinet. Clinical samples were extracted using one of two automatic nucleic acid isolation systems according to their availability in different MOH-SA regional laboratories including: MagNA Pure 96 (Roche Diagnostics, Indianapolis, IN) and QIAsymphony SP (Qiagen, Hilden, Germany). MagNA Pure Compact Nucleic Acid Isolation (Roche) and QIAsymphony RNA (Qiagen) kits were utilized for viral RNA extraction in the corresponding platform according to the manufacturer's instructions. Single negative control of PCR grade water was extracted in parallel for every 12 samples. All samples were screened for MERS-CoV using LightMix® Modular MERS-CoV upE Kit (Roche) using the experimental protocol and reaction setup primarily established by Corman et al. [28 (link)]. Positive results were confirmed with LightMix® Modular MERS-CoV ORF1a Kit (Roche). Samples with doubtful results (i.e. positive reactivity in one assay and negative in the other) were re-extracted and tested in triplicates in both UpE and ORF1a assays.
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