Genechip 3 ivt plus reagent kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan

The GeneChip 3' IVT PLUS Reagent Kit is a tool used in the process of gene expression analysis. It is designed to generate labeled and amplified complementary RNA (cRNA) from messenger RNA (mRNA) samples. The kit provides the necessary reagents and protocols to enable this key step in the gene expression profiling workflow.

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Plus Reagent (704 citations) GeneChips (22 citations) 3’ IVT Plus Kit (13 citations) GeneChip 3′IVT PLUS Kit (7 citations) GeneChip™ HT 3′IVT PLUS Reagent Kit (6 citations) 3′ IVT PLUS Reagent Kit (4 citations) PLUSTM (3 citations) Human GeneChip 3′ IVT PLUS Reagent Kit (3 citations) GeneChip® 3′ Expression Arrays and 3′ IVT PLUS Reagent Kit (3 citations) GeneChip 3’ IVT Reagent Kit (2 citations)

55 protocols using genechip 3 ivt plus reagent kit

Profiling TGF-β Signaling Pathways

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The second step was associated with evaluation of the expression pattern of mRNAs related to TGFβ paths using expression microarray HG-U133A 2.0 (Affymetrix, Santa Clara, CA) and GeneChip™ 3′ IVT PLUS Reagent Kit and GeneChip™ HT 3′ IVTPLUS Reagent Kit (Thermo Fisher, catalog number 902416) according to the manufacturer's protocol.
The names of probes and number of their ID were found from the Affymetrix NetAffx™ Analysis Center database after entering the phrase “TGF beta signaling pathways” (http://www.affymetrix.com/analysis/index.affx; accessed on 10^th March 2020).
The microarray analysis consisted of synthesizing cDNA from RNA extracts, adding poly-A control, synthesizing of biotinyl aRNA and hybridization aRNA of specimens with microarray probes and data analysis. The detailed protocol for molecular analysis was also described in our previous studies [37 (link), 38 (link)].

Adwent I., Grabarek B.O., Kojs-Mrożkiewicz M., Brus R., Staszkiewicz R., Plewka A., Stasiowski M, & Lyssek-Boroń A. (2020). The Influence of Adalimumab and Cyclosporine A on the Expression Profile of the Genes Related to TGFβ Signaling Pathways in Keratinocyte Cells Treated with Lipopolysaccharide A. Mediators of Inflammation, 2020, 3821279.

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Endothelin Expression Profiling via Microarray

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Expression profile of endothelins and their receptors was determined using the GeneChip 3′IVT PLUS Reagent Kit (ThermoFisher Scientific, Waltham, MA, USA) and HG-U133A 2_0 oligonucleotide microarrays (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. Fluorescence signals were acquired by the GeneArray scanner (Agilent Technologies, Santa Clara, CA, USA). The methodology has been described in detail in other articles (Grabarek et al. 2021 (link)). Microarray experiment was further validated with RT-qPCR.

Zmarzły N., Januszyk S., Mieszczański P., Morawiec E., Buda P., Dziobek K., Opławski M, & Boroń D. (2022). Endothelin-3 is epigenetically silenced in endometrioid endometrial cancer. Journal of Cancer Research and Clinical Oncology, 149(9), 5687-5696.

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Microarray Analysis of Cln3 Mutant Brain Tissue

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Microarray analysis of 16 Cln3^Δex7/8 brain tissue samples treated with vehicle or GalCer (four males and four females in each group) was performed using the GeneChip Mouse Genome 430 2.0 array platform from Affymetrix (Affymetrix Inc., CA, USA) representing over 45,000 probe sets) (Fig 1). Samples were prepared and microarrays processed using the GeneChip™ 3’ IVT PLUS Reagent Kit (Thermo Fisher Scientific, MA, USA) according to manufacturer instructions. Briefly, 100 ng of total RNA were labeled, fragmented, and then hybridized to arrays. After washing and staining using GeneChip Fluidics Station 450 (Thermo Fisher Scientific, MA, USA), arrays were scanned with the GeneChip Scanner 3000 7G (Thermo Fisher Scientific, MA, USA). Cell Intensity Data (CEL) files were generated with the Affymetrix GeneChip™ Command Console (AGCC) software version 3.2 (Thermo Fisher Scientific, MA, USA).

Makoukji J., El-Sitt S., Makhoul N.J., Soueid J., Kadara H, & Boustany R.M. (2020). Sex differences in gene expression with galactosylceramide treatment in Cln3Δex7/8 mice. PLoS ONE, 15(10), e0239537.

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Microarray Analysis of Head and Neck SCC Transcriptome

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Microarray analysis was performed as follows. Total RNA was extracted by lysing the tissues with Isogen (Nippon Gene) following homogenization with a TissueLyser (Qiagen AB). Total RNA (500 ng) was used to generate biotin-labeled cRNA using a GeneChip^® 3′ IVT PLUS Reagent kit (Thermo Fisher Scientific, Inc.). The biotin-labeled RNA was hybridized to Human Genome U-219 Array Strips (Thermo Fisher Scientific, Inc.). After washing and staining the strips, the signal was developed and scanned using GeneAtlas (Thermo Fisher Scientific, Inc.). Data analysis was conducted using GeneSpring GX 14.9.1 (Agilent Technologies GmbH). The robust multichip average method was used with background correction and normalization. Fold change analysis was performed to identify genes with >3-fold differences, using a moderated t-test (P<0.05) with Benjamini-Hochberg multiple correction. Gene expressions of KRT8, ABCC3, GCLC, RYBP, TMEM97, SLC1A3, IRS2, KYNU, CSAG2, CDA and CCL21 in head and neck SCCs (528 cases) from the cancer genome atlas were analyzed by OncoPrint from cBioPortal. Microarray data have been deposited in the Gene Expression Omnibus (GEO; experiment. no. GSE153918) database using minimum information about microarray experiment guidelines. The algorithm of the clustering method for the microarray data is shown in Fig. S1.

Hasegawa T., Nakashiro K.I., Fukumoto C., Hyodo T., Sawatani Y., Shimura M., Kamimura R., Kuribayashi N., Fujita A., Uchida D, & Kawamata H. (2021). Oral squamous cell carcinoma may originate from bone marrow-derived stem cells. Oncology Letters, 21(2), 170.

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Microarray Analysis of Gene Expression

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For microarray analysis, 100 ng total RNA was amplified using the GeneChip 3' IVT Plus Reagent kit (Thermo Fisher Scientific) generating biotinylated complementary RNA. The labeled samples were hybridized to GeneChip HT MG-430 PM arrays (Thermo Fisher Scientific). Washing, staining, and scanning was performed using the GeneTitan Wash, Stain Kit for 3' IVT Array Plates, and the GeneTitan Instrument (Thermo Fisher Scientific). Data were analyzed using the R2 Genomics analysis and visualization platform (http://r2.amc.nl).

Kavazović I., Han H., Balzaretti G., Slinger E., Lemmermann N.A., ten Brinke A., Merkler D., Koster J., Bryceson Y.T., de Vries N., Jonjić S., Klarenbeek P.L., Polić B., Eldering E, & Wensveen F.M. (2020). Eomes broadens the scope of CD8 T-cell memory by inhibiting apoptosis in cells of low affinity. PLoS Biology, 18(3), e3000648.

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Transcriptome Profiling of Cell Types

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Quality and quantity of the 60 nucleic acid samples were established using the Agilent Bioanalyzer RNA Nano kit and the Nanodrop 1000, respectively. GeneChip 3′ IVT PLUS Reagent Kit (Thermo Scientific) provided sufficient amplified double-stranded cDNA for each holoclone, meroclone, or paraclone total RNA sample, letting to in vitro transcribe it to labeled cRNA, that could be fragmented and therefore hybridized onto single GeneChip™ Human Genome U133 Plus 2.0 Array (ThermoScientific), following the manufacturer’s indications. To avoid batch effect among samples, i.e. no technical sources of variation added to the samples during handling, samples were randomized during RNA isolation, sample preparation, and hybridization/scanning working sessions. All samples were processed with the same reagents lot number, when available. Sample and hybridization quality controls were carried out with Transcriptome Analysis Console (TAC, ThermoScientific) to verify complete and unbiased coverage of the transcriptome.

Enzo E., Secone Seconetti A., Forcato M., Tenedini E., Polito M.P., Sala I., Carulli S., Contin R., Peano C., Tagliafico E., Bicciato S., Bondanza S, & De Luca M. (2021). Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells. Nature Communications, 12, 2505.

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Microarray Analysis of Drug Resistance

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The microarray profile of mRNA expression changes that are associated with drug resistance was determined using the HG-U133A 2_0 microarray (Affymetrix, Santa Clara, CA, USA), the GeneChip™ 3′ IVT PLUS Reagent Kit, and GeneChip™ HT 3′ IVT PLUS Reagent Kit (Thermo Fisher Scientific, Waltham, MA USA, Cat. no. 902416) according to the manufacturer’s recommendations. The mRNA names and their ID number were determined from the Affymetrix NetAffx™ Analysis Center database after entering the phrase “drug resistance” (http://www.affymetrix.com/analysis/index.affx; accessed on February 2, 2022). Data were analyzed using a microarray scanning GeneArray scanner (Agilent Technologies, Santa Clara, CA, USA).

Opławski M., Średnicka A., Niewiadomska E., Boroń D., Januszyk P, & Grabarek B.O. (2022). Clinical and molecular evaluation of patients with ovarian cancer in the context of drug resistance to chemotherapy. Frontiers in Oncology, 12, 954008.

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Gene Expression Profiling of Pluripotent Cells

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Biotinylated cRNA was prepared from 100 ng of total RNA using a GeneChip 3'IVT PLUS Reagent kit (ThermoFisher Scientific, Waltham, MA) and was hybridized to the GeneChip PrimeView Human Gene Expression Array (ThermoFisher Scientific) for 16 hours at 45°C. Array slides were washed and stained in an Affymetrix Fluidics station 450, and scanned using a GeneChip scanner 3000 7G system. The data files were then uploaded to www.pluritest.org, scored for pluripotency, and deposited into the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database with accession no. GSE104605.

Watanabe D., Koyanagi‐Aoi M., Taniguchi‐Ikeda M., Yoshida Y., Azuma T, & Aoi T. (2017). The Generation of Human γδT Cell‐Derived Induced Pluripotent Stem Cells from Whole Peripheral Blood Mononuclear Cell Culture. Stem Cells Translational Medicine, 7(1), 34-44.

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EMT Gene Expression Profiling

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The evaluation of the expression pattern of mRNAs connected with the EMT was done using oligonucleotide microarray microarrays HG-U133A 2_0 (Affymetrix, Santa Clara, CA, USA), the GeneChip™ 3′IVT PLUS Reagent Kit, and GeneChip™ HT 3′IVT PLUS Reagent Kit (ThermoFisher, Catalog Number 902416) according to the manufactures’ protocol. The names of the probes and their ID number were obtained from the Affymetrix NetAffx™ Analysis Center database after entering the phrase “EMT” (http://www.affymetrix.com/analysis/index.affx; accessed on 25 November 2020). Data were analyzed via using microarray scanning GeneArray scanner (Agilent Technologies, Santa Clara, CA, USA).

Opławski M., Nowakowski R., Średnicka A., Ochnik D., Grabarek B.O, & Boroń D. (2021). Molecular Landscape of the Epithelial–Mesenchymal Transition in Endometrioid Endometrial Cancer. Journal of Clinical Medicine, 10(7), 1520.

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Biogenic Amine Gene Expression Profiling

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The biogenic amine-related gene expression profile was determined using HG-U133A 2.0 oligonucleotide microarrays (Affymetrix, Santa Clara, CA, USA), the GeneChip™ 3′IVT PLUS Reagent Kit (ThermoFisher, Waltham, MA, USA, Cat. No. 902416), and the GeneChip™ HT 3′IVT PLUS Reagent Kit (ThermoFisher, Waltham, MA, USA, Cat. No. 902417).
The names of the probes and their identification numbers were obtained by entering the phrase “biogenic amine” in the Affymetrix NetAffx™ Analysis Center database (http://www.affymetrix.com/analysis/index.affx; accessed on 1 July 2021). Gene Array Scanner (Agilent Technologies, Santa Clara, CA, USA) was used to measure the fluorescence intensity.

Czerwiński M., Bednarska-Czerwińska A., Zmarzły N., Boroń D., Oplawski M, & Grabarek B.O. (2021). Evaluation of the Differences in the Expression of Biogenic Amine-Related mRNAs and Proteins in Endometrioid Endometrial Cancer. Journal of Clinical Medicine, 10(21), 4872.

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