Genechip human transcriptome array 2 hta 2

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The GeneChip® Human Transcriptome Array 2.0 (HTA 2.0) is a genome-wide array designed to analyze the expression of protein-coding and non-coding transcripts. It provides comprehensive coverage of the human transcriptome.

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17 protocols using genechip human transcriptome array 2 hta 2

Gene Expression Profiling of Whole Blood

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RNA was extracted from whole blood collected in Tempus tubes. Gene expression was assessed using the GeneChip® Human Transcriptome Array 2.0 (HTA 2.0) (Affymetrix, USA) at the University of Santiago de Compostela (USC, Spain). Samples were randomized and balanced by sex and cohort within each batch. Data was normalized at the gene level with the GCCN (SST-RMA) algorithm, and batch effects and blood cell type composition were controlled with two surrogate variable analysis (SVA) methods, isva [38 (link)] and SmartSVA [39 (link)], during the differential expression analyses. Gene expression values were log2 transformed, and annotation of transcript clusters (TCs) to genes was done with the Affymetrix Expression Console software using the HTA-2_0 Transcript Cluster Annotations Release na36 (hg19).

Vives-Usano M., Hernandez-Ferrer C., Maitre L., Ruiz-Arenas C., Andrusaityte S., Borràs E., Carracedo Á., Casas M., Chatzi L., Coen M., Estivill X., González J.R., Grazuleviciene R., Gutzkow K.B., Keun H.C., Lau C.H., Cadiou S., Lepeule J., Mason D., Quintela I., Robinson O., Sabidó E., Santorelli G., Schwarze P.E., Siskos A.P., Slama R., Vafeiadi M., Martí E., Vrijheid M, & Bustamante M. (2020). In utero and childhood exposure to tobacco smoke and multi-layer molecular signatures in children. BMC Medicine, 18, 243.

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Transcriptomics of Monozygotic Twin Siblings

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Patients’ data regarding disease presentation and its clinical and radiological course were collected from medical records updated at each follow-up visit. Patients provided informed consent and ethical approval was granted by the local NHS authority (PROMISES/AC/2018 Prot. PG/2018/16313), in accordance with the 1964 Helsinki declaration.
The transcriptomic analysis was carried out using the mRNAs from whole blood (Qiagen RNA blood column kit) of the couple of MZ. Then, prior to the gene expression study, the RNA integrity and quality were checked out by Bioanalyzer (Agilent) and transcripts were analyzed by GeneChip Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix); the full technical report is available in

Supplementary Material

. Transcripts differentially expressed between the two brothers were analyzed by the commercial software Partek Genomics Suite V 6.6, values are reported by a Fold Change cutoff ± 2.0 (further technical details and full transcripts’ list are available in

Supplementary Data

). Signal pathways and pathophysiological processes in which the abovementioned mRNAs are eventually involved, were in silico analyzed by the Ingenuity Pathway Analysis (IPA) software (QIAGEN Inc.,

https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis

Angioni M.M., Floris A., Cangemi I., Congia M., Chessa E., Orrù S., Piga M, & Cauli A. (2021). Gene Expression Profiling of Monozygotic Twins Affected by Psoriatic Arthritis. Open Access Rheumatology : Research and Reviews, 13, 23-29.

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Transcriptome Profiling of Liver Disease

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Total RNA was isolated from the liver biopsy samples, using the RNeasy kit (Invitrogen life technologies, Carlsbad, CA), according to the manufacturer’s protocol, and a new high-resolution array from Affymetrix, GeneChip Human Transcriptome Array 2.0 (HTA2.0), was undertaken at the UTSWMC core facility to interrogate all transcript isoforms in the human transcriptome with 6 million probes targeting coding transcripts, exon-exon splice junctions, and non-coding transcripts to identify molecular signatures in each of the early and advanced stages of liver disease from HCV mono- and HIV-1/HCV-co-infected liver samples. To evaluate the microarray data, we employed one-way between-subject ANOVA (unpaired) and ANOVA p-value (condition pair) analysis wherein a p-value of < 0.05 was considered statistically significant (*). Fold change (linear) <-2 or fold change (linear) >2 was used as default filter criteria.

Whitmill A., Kim S., Rojas V., Gulraiz F., Afreen K., Jain M., Singh M, & Park I.W. (2018). Signature molecules expressed differentially in a liver disease stage-specific manner by HIV-1 and HCV co-infection. PLoS ONE, 13(8), e0202524.

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Gene Expression Profiling of Immune Responses

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To obtain unbiased results, we created pooled RNA libraries by evenly pooling six RNA samples, which resulted in three pooled healthy control, three fever control, three pre-IVIG, and three post-IVIG libraries, as previous described 9 (link). We performed microarray assay on the pooled RNA samples to establish the gene expression profiles and then further performed profiling with GeneChip® Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix, Santa Clara). We used the WT PLUS Reagent kit to prepare the RNA samples and carry out hybridization on the HTA 2.0 microarray chips. Adhering to the Affymetrix instruction manual, we subjected the HTA 2.0 chips' raw data to quality control examination, as previously described in another study 9 (link), 15 (link).

Huang Y.H., Chen K.D., Lo M.H., Cai X.Y., Chang L.S., Kuo Y.H., Huang W.D, & Kuo H.C. (2019). Decreased DNA methyltransferases expression is associated with coronary artery lesion formation in Kawasaki disease. International Journal of Medical Sciences, 16(4), 576-582.

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Transcriptome Analysis of Cell Samples

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Approximately 1 × 10⁷cells were collected and treated with Trizol reagent (Life Technologies) in an RNase-free tube. The RNA isolation from the cells, microarray analysis, data processing, statistical analysis and gene ontology analysis were performed by the Shanghai Biotechnology Corporation, Shanghai, China. The microarray experiments were performed using the Affymetrix GeneChip Human Transcriptome Array 2.0 (HTA2.0).

Wu Y., Liu M., Li Z., Wu X.B., Wang Y., Wang Y., Nie M., Huang F., Ju J., Ma C., Tan R., Zen K., Zhang C.Y., Fu K., Chen Y.G., Wang M.R, & Zhao Q. (2015). LYAR promotes colorectal cancer cell mobility by activating galectin-1 expression. Oncotarget, 6(32), 32890-32901.

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CLL Cell RNA Isolation and Microarray Analysis

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Total RNA enriched in small RNA (>200 bp) was isolated from primary CLL cells following drug treatment using miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the supplier’s protocol. Total RNA concentration was measured by using Nano Drop UV/Vis spectrophotometer (2000c, Thermo Scientific). RNA quality was assessed by capillary electrophoresis with a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Afterwards, the RNA was pooled (2 μg/ patient sample), creating 3 pools per treatment group. Prior to hybridization, RNA integrity and comparability were tested by BioAnalyzer and only samples with RNA integrity numbers (RIN) > 8.0 were used in microarray experiments. cDNA synthesis, labelling and the basic microarray analysis was performed by the Genomics Shared Resource at The Ohio State University Comprehensive Cancer Center. Generation of double-stranded cDNA, preparation and labelling of cRNA, hybridization to GeneChip® Human Transcriptome Array 2.0 (HTA 2.0) (Affymetrix, Santa Clara, CA, USA) and washing was performed according to the standard Affymetrix protocol. The arrays were scanned using a GeneChip® Scanner 3000 (Affymetrix). Signal intensities were analyzed by Affymetrix Expression Console software.

Ozer H.G., El-Gamal D., Powell B., Hing Z.A., Blachly J.S., Harrington B., Mitchell S., Grieselhuber N.R., Williams K., Lai T.H., Alinari L., Baiocchi R.A., Brinton L., Baskin E., Cannon M., Beaver L., Goettl V.M., Lucas D.M., Woyach J.A., Sampath D., Lehman A.M., Yu L., Zhang J., Ma Y., Zhang Y., Spevak W., Shi S., Severson P., Shellooe R., Carias H., Tsang G., Dong K., Ewing T., Marimuthu A., Tantoy C., Walters J., Sanftner L., Rezaei H., Nespi M., Matusow B., Habets G., Ibrahim P., Zhang C., Mathé E.A., Bollag G., Byrd J.C, & Lapalombella R. (2018). BRD4 profiling identifies critical Chronic Lymphocytic Leukemia oncogenic circuits and reveals sensitivity to PLX51107, a novel structurally distinct BET inhibitor. Cancer discovery, 8(4), 458-477.

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Transcriptomic Analysis of Kawasaki Disease

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As previous reports have described [23 (link)], total mRNA samples from 18 healthy controls, 18 febrile controls, and 18 KD patients 24 h before IVIG was given (KD1 group), and 18 KD patients 21 days after IVIG therapy (KD2 group) were pooled together into three RNA libraries, each containing RNA samples from 6 patients. All RNA samples were then prepared for hybridization to the GeneChip^® Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix, Santa Clara) using the WT PLUS Reagent kit. Hybridized HTA 2.0 microarray chips were checked for quality, and the gene expression data was then analyzed with commercially available software (Partek, St. Louis, MI, USA).

Huang Y.H., Chen K.D., Kuo K.C., Guo M.M., Chang L.S., Yang Y.L, & Kuo H.C. (2022). Human Transcriptome Array Analysis Identifies CDR2 as a Novel Suppressed Gene for Kawasaki Disease. Diagnostics, 12(2), 240.

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Macrophage Polarization Profiling in Kawasaki Disease

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For unbiased results, 18 mRNA samples each from the healthy control group (HC group), KD1 group (KD patients 24 h prior to IVIG therapy) and the KD2 group (KD patients 21 days after IVIG therapy) were combined into three pooled RNA libraries each containing six RNA samples. We then used GeneChip® Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix, Santa Clara) to determine gene expression profiles of macrophage surface markers which are commonly expressed on M1 and M2 macrophages after literature review (13 (link)–16 (link)). Prior to hybridization to the HTA 2.0 microarray chips, all RNA samples were prepared according to manufacturer instructions with the WT PLUS Reagent kit. The hybridized HTA 2.0 microarray chips then underwent quality control inspection. All chips passed quality control examination and were then analyzed using commercial software specific for microarray data analysis (Partek, St. Louis).

Guo M.M., Chang L.S., Huang Y.H., Wang F.S, & Kuo H.C. (2020). Epigenetic Regulation of Macrophage Marker Expression Profiles in Kawasaki Disease. Frontiers in Pediatrics, 8, 129.

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Microarray Gene Expression Profiling

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For strong, unbiased results, pooled RNA libraries were created by evenly pooling six RNA samples, which resulted in three pooled normal control, three fever control, three pre-IVIG, and three post-IVIG libraries. We submitted the pooled RNA samples to microarray assay to establish the gene expression profiles. We further performed profiling with GeneChip^® Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix, Santa Clara). We used the WT PLUS Reagent kit to prepare the RNA samples and then performed hybridization on the HTA 2.0 microarray chips. In accordance with the Affymetrix instruction manual, the raw data of the HTA 2.0 chips underwent quality control examination, as previously described [13 (link)].

Kuo H.C., Li S.C., Huang L.H, & Huang Y.H. (2017). Epigenetic hypomethylation and upregulation of matrix metalloproteinase 9 in Kawasaki disease. Oncotarget, 8(37), 60875-60891.

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Transcriptome Analysis of rFGL2 Treatment

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RNA samples were obtained after the treatment of MDM (4 different healthy donors) with rFGL2 using GeneChip Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix). Microarray data were analyzed using the Gene-Cloud of Biotechnology Information (GCBI) platform. To identify genes affected by rFGL2 by Gene Ontology analysis, genes were selected with a minimum fold change of 1.5 and based on an adjusted P value < 0.05.

Hou X.X., Wang X.Q., Zhou W.J, & Li D.J. (2021). Regulatory T cells induce polarization of pro-repair macrophages by secreting sFGL2 into the endometriotic milieu. Communications Biology, 4, 499.

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