Transcriptome analysis console software 4

Total RNA was extracted from the frozen gastrocnemius muscle of recipient mice using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Target hybridization and cRNA preparations were performed according to the Affymetrix GeneChip® Technical Protocol (Affymetrix, Santa Clara, CA, USA). Affymetrix GeneChip® Mouse Gene 1.0 ST arrays were stained and washed in an Affymetrix Fluidics Station 450 and scanned using a GeneChip® Scanner 3000 7G (Affymetrix). Expression levels were analyzed using Transcriptome Analysis Console Software 4.0 (Affymetrix) following background correction, signal summarization, and normalization by SST[EMS1]-RMA. Pathways that were significantly enriched in the list of differentially expressed genes were identified using Ingenuity® Pathway Analysis (IPA®, QIAGEN).

Microarray analysis was performed as described previously [13 (link)]. Briefly, a GeneAtlas® system (Affymetrix, Santa Clara, CA, USA) was employed. RNA extraction and quality control were performed as described previously [13 (link)]. Labeled fragmented single-stranded cDNA (ss-cDNA) was synthesized by using purified total RNA (100-500 ng) as the template following Affymetrix WT PLUS Labeling Assay protocol. Then, the mixtures of biotinylate labeled ss-cDNAs were hybridized onto porcine Gene 1.1 ST Arrays (Affymetrix, Santa Clara, CA, USA). After being washed by a fluidic station, the arrays were scanned with an imaging station in a GeneAtlas® system (Affymetrix, Santa Clara, CA, USA), and the acquired array raw data were analyzed with the Affymetrix Command Console Software Version 1.4. Quantile normalization and subsequent data processing were performed by the Affymetrix Transcriptome Analysis Console (TAC) Software 4.0. The cutoffs for differentially expressed genes (DEGs) were set at fold change > 1.5 or <0.67 and FDR < 0.01. Gene Ontology (GO) (http://www.geneontology.org/) and Reactome pathway database (http://www.reactome.org) were used for further analysis.

RNA from isolated PBMC was extracted using TRIzol Reagent (Thermo Fisher Scientific). RNA from 4 TRDC-knockout pigs and 2 wild type pig were pooled, respectively. Pools were used to assess the quality of the RNA using an agilent bioanalyser. Labeled fragmented single-stranded cDNAs (ss-cDNA) were synthesized by using purified total RNA (100–500 ng) as template following Affymetrix WT PLUS Labeling Assay protocols. Porcine Gene 1.1 ST Arrays (Affymetrix, Santa Clara, CA, USA) were hybridized to the biotinylated ss-cDNA targets. After 20 h of hybridization at 48 °C, arrays were washed by a fluidics station and then scanned by an imaging station in a GeneAtlas System (Affymetrix, Santa Clara, CA, USA). After scanning, the intensity data (CEL files) of Porcine Gene 1.1 ST Arrays (Affymetrix) were extracted from the image data (DAT files) by the Affymetrix Command Console Software Version 1.4, and then normalized and analyzed by the Affymetrix Transcriptome Analysis Console (TAC) Software 4.0 for gene expression profiles and DEGs. The DEGs were selected by a cutoff of fold change > 2.

The raw data were normalized in Expression Console, provided by Affymetrix (http://www.affymetrix.com, accessed on 23 February 2016), using the robust multiarray average (RMA) method that was first suggested by Li and Wong [16 (link),17 (link)]. Subsequent analysis of the differences in the gene expression was carried out in the Transcriptome Analysis Console (TAC) Software 4.0 (Affymetrix Inc., Santa Clara, CA, USA). To increase the percentage of variance and to better visualize the patterns of the original dataset, a 3D Principal Component Analysis (PCA)-plot was used to visualize the microarray data.

RNA was isolated from 3 snap frozen GD 9.5 implantation sites randomly chosen from each of 3 WT and 3 IL15Δ/Δ rats at GD 9.5 (a total of 9 implantation sites per group). RNA was extracted by homogenizing whole implantation sites in RiboZol (VWR), followed by collection of the aqueous phase which was applied to RNeasy columns (Qiagen). Following DNase I (Qiagen) treatment, purified RNA (30 µg per sample) from the 3 implantation sites per dam were combined to generate 3 samples per group, and samples were submitted to Hamilton Health Sciences Centre (Hamilton, ON, Canada) for transcriptome analysis on a GeneChip Scanner 3000 using the Clariom S rat array (ThermoFisher Scientific), as previously described (Roberts et al., 2021 (link)). Samples were normalized using the SST-RMA (Signal Space Transformation- Robust Multiarray Analysis) data normalization algorithm to reduce background. Data files were generated and processed for analysis using Transcriptome Analysis Console Software 4.0 (ThermoFisher Scientific) to analyze global gene expression patterns. Gene ontology pathway analysis was completed using DAVID Functional Annotation Bioinformatics (Huang et al., 2009 (link)).

The COVID group was compared with the control group using Transcriptome Analysis Console software 4.0 (TAC 4.0, Thermo Fisher Scientific, Waltham, MA). Sst-RMA normalization was performed on 16 cel files generated from the samples. Student t-test was performed for comparison of the two groups. Genes with fold change ≥1.5 and p ≤ 0.05 were identified as differentially expressed. Gene expression data is available at the Gene Expression Omnibus (GEO) database of the NIH (Accession number GSE195938). Data were analyzed through the use of IPA (QIAGEN Inc., IPA">https://digitalinsights.qiagen.com/IPA) (23 (link)). IPA is one of advanced bioinformatic tools provided by Qiagen Inc. that is a web-based software application program for the analysis, integration, and interpretation of data derived from microarray, gene expression or other array based or sequencing methods. IPA analyses and interprets data based on the comprehensive, manually curated content of the Ingenuity Knowledge Base. IPA identifies Canonical pathways, Networks, Tox Functions and Upstream regulators.