Geneatlas imaging station

Aliquots (100 ng) of total RNA obtained from 3 animals per group on week 9 (cohort 1) and 4 animals per group on weeks 17 (cohort 2) and 22 (cohort 3) were individually converted to cRNA and labeled with a Gene ChIP® Poly-A RNA Control Kit, WT Amplification Kit, and Gene ChIP® WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. Hybridization, washing, and staining were performed using Affymetrix® MoGene2.1 ST Array Strips and a GeneAtlas® Hybridization Wash and Stain Kit for WT Assay Strips (Affymetrix), according to the manufacturer’s protocols. After washing, MoGene2.1 Array Strips were analyzed using a GeneAtlas Imaging Station (Affymetrix). Data analyses were performed using Expression Console (Affymetrix) and Transcriptome Analysis Console (Affymetrix). The cut-off point: ≤−2 or ≥2 of a linear fold change and ANOVA P values were used as described in our previous studies^{45 (link),46 (link)}.

Biotin-labelled and fragmented target cRNA samples were loaded into Affymetrix GeneChip (Human Genome U219) Array Strips together with controls cRNAs and oligo B2. The hybridisation procedure was conducted at 45°C for 16 h in an AccuBlock Digital Dry Bath (Labnet international, Inc.) hybridisation oven. The washing and staining procedure was performed using an Affymetrix GeneAtlas Fuidics Station according to the instructions in the technical manual. An Affymetrix GeneAtlas Imaging Station was used for scanning the arrays.

To conduct microarray experiments, 100 ng of total RNA of each condition was used as input. Samples were reversely transcribed into cDNA and then in vitro transcribed according to the manufacturer’s protocol using GeneChip™ 3′ IVT PLUS Reagent Kit (Applied Biosystems, Thermo Fisher Scientific Inc.). Next, samples were fragmented and hybridized on Affymetrix GeneChip human genome U219 microarrays, together with control cRNA and oligo B2. Hybridization was conducted at 45 °C for 16 h, using an AccuBlock™ Digital dry bath (Labnet International, Inc., New York, NY, USA) hybridization oven. Further, the microarrays were washed and stained according to the manufacturer’s protocol using an Affymetrix GeneAtlas™ Fluidics Station (Affymetrix, Santa Clara, CA, USA). In the final step, all microarrays were scanned using an Affymetrix GeneAtlas™ imaging station (Affymetrix). The scans of the microarrays were saved as *.CEL files on local storage. Downstream analysis was conducted in R/RStudio IDE (R – version 4.0.2, RStudio – version 1.3.1056), Bioconductor (version 1.3.10) as well as Transcriptome Analysis Software 4.0.1.36. Microarray results were stored in the GEO database under ID GSE140996.

Total RNA was isolated from oxyntic and PYL collected from each subject, according to Qiagen RNeasy Midi Kit protocol (Qiagen, Hilden, Germany). To reduce the viscosity of the lysate, specimens (50 to 100 mg) were homogenized directly in the buffer RTL containing guanidine thiocyanate. All the other procedures were in agreement with the manufacturer protocol. Purity and integrity evaluation was assessed just before analysis by Agilent Bioanalyzer 2100. Total RNA was hybridized on Affymetrix Porcine Gene 1.1 ST array strips. Hybridized arrays were scanned on a GeneAtlas imaging station (Affymetrix, Santa Clara, CA, USA). Performance quality tests of the arrays including the labelling, hybridization, scanning and background signals by a Robust Multichip Analysis were performed on the CEL files using Affymetrix Expression Console. The intensity records were log2-transformed. Transcript data have been submitted to the National Center for Biotechnology Information's Gene Expression Omnibus (NCBI GEO) with GEO accession number GSE57620.

Total RNA was isolated from the gastric tissue samples using the FastPure RNA kit (TaKaRa Bio Inc., Shiga, Japan). All other procedures were in agreement with the manufacturer’s protocol. The RNA purity and integrity were evaluated using Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA) just before the transcriptome analysis. The total RNA was hybridized on Affymetrix Porcine Gene 1.1 ST array strips. The hybridized arrays were scanned on a GeneAtlas imaging station (Affymetrix, Santa Clara, CA, USA). Performance quality tests of the arrays including the labelling, hybridization, scanning and background signals using Robust Multichip Analysis were carried out on the CEL files using the Affymetrix Expression Console.

Microarray hybridization probes were generated from isolated RNA extracted from the skin treated with vehicle or TCQA using ISOGEN solution (Nippon Gene, Tokyo, Japan) following the manufacturer’s instructions. Briefly, the extracted RNA was amplified and biotin labeled as aRNA. Then, the fragmented, biotin-labeled aRNA was hybridized to the Affymetrix mouse 430 PM Array strips containing probes for 45,141 mouse genes. Hybridized arrays were washed and stained in GeneAtlas Fluidics Station. The GeneChip (Mouse Genome 430 2.0 Array) was scanned using the Affymetrix GeneAtlas Imaging Station to obtain the mRNA expression data of genes from mouse genome. The generated data (twofold change, control vs. TCQA) were then analyzed using Transcriptome Analysis Console (TAC) Software (version 4.0.1) and database for annotation, visualization, and integrated discovery (DAVID) bioinformatics resources 6.8 (Huang et al., 2009 (link); Zacariotti et al., 2010 (link)).

Total RNA (130 ng) enriched for low molecular weight RNA from each sample was labelled using the FlashTag Biotin HSR RNA Labelling Kit (Affymetrix) on the GeneAtlas Hybridization Station (Affymetrix) and subsequently, it was processed using the GeneAtlas Hybridization, Wash and Stain Kit for miRNA Array Strips (Applied Biosystems, USA) on the GeneAtlas Fluidics Station (Affymetrix) according to the manufacturer’s instructions. Array strip fluorescence intensities were finally determined using the GeneAtlas Imaging Station (Affymetrix). Raw data were processed and visualized using Partek Genomics Suite software (Partek, USA).