Genechip microrna 4.0 array

Manufactured by Thermo Fisher Scientific

Sourced in United States

The GeneChip microRNA 4.0 arrays from Thermo Fisher Scientific are a comprehensive solution for the detection and quantification of microRNA expression profiles. The arrays provide comprehensive coverage of human, mouse, and rat microRNA sequences from miRBase, a comprehensive database of published microRNA sequences and annotation. The arrays offer a high-density format, enabling the analysis of multiple samples simultaneously.

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9 protocols using genechip microrna 4.0 array

Microarray Analysis of miRNA Expression

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Total RNA was extracted using the TRIzol Reagent (Invitrogen, Carlsbad, CA USA) according to the manufacturer’s instructions. RNA quality and quantity was assessed by Agilent bioanalyzer 2100 analysis (Agilent Technologies, CA, USA).
Starting with 250 ng of total RNA, the labeling process begins with poly-A tailing of each RNA strand using poly-A polymerase, followed by ligation of biotin-labeled 3DNA dendrimer. Biotinylated RNA strands were hybridized at 48 °C for 18 h on the GeneChip microRNA 4.0 Array (Affymetrix, Santa Clara, CA, USA). The GeneChip microRNA 4.0 Array was washed and stained in the Fluidics Station 450 (Affymetrix, CA, USA). Amplified fluorescence signals by the branched structure of 3DNA dendrimer were scanned using GeneChip Scanner 3000 7G (Affymetrix).
The arrays were analyzed using an Agilent scanner with associated software. The expression levels of miRNAs were calculated with Expression Console 1.4 (Affymetrix, Santa Clara, CA, USA). Relative signal intensities for each miRNA were generated using the Robust Multi-Array Average algorithm. Target prediction were analyzed using the miRBase, miRDB, TargetScan and microRNA.org DB.

Lee S.H., Ju H.M., Choi J.S., Ahn Y., Lee S, & Seo Y.J. (2018). Circulating Serum miRNA-205 as a Diagnostic Biomarker for Ototoxicity in Mice Treated with Aminoglycoside Antibiotics. International Journal of Molecular Sciences, 19(9), 2836.

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Microarray-based profiling of miRNA expression

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Microarrays were processed according to manufacturer's recommendations. For each sample, 10 μL of RNAs were labeled with FlashTag™ Biotin HSR RNA Labeling Kit (Genisphere – Affymetrix^® UK Ltd, High Wycombe, United Kingdom). Samples were hybridized for 16 h at 48 °C on GeneChip^® microRNA 4.0 Array (Affymetrix^®) and scanned with a GC30007G scanner (Affymetrix^®). Raw data were normalized using the Expression Console (Affymetrix^®) with the RMA method, algorithmically based on microarrays spike‐in and called the « standard normalization ». The additional « exogenous normalization » was achieved for each sample by dividing all human miRNAs intensities by the geometrical mean of the three cel‐miRs intensities. Data have been deposited in the NCBI Gene Expression Omnibus database (GSE69341).

Vigneron N., Meryet-Figuière M., Guttin A., Issartel J.P., Lambert B., Briand M., Louis M.H., Vernon M., Lebailly P., Lecluse Y., Joly F., Krieger S., Lheureux S., Clarisse B., Leconte A., Gauduchon P., Poulain L, & Denoyelle C. (2016). Towards a new standardized method for circulating miRNAs profiling in clinical studies: Interest of the exogenous normalization to improve miRNA signature accuracy. Molecular Oncology, 10(7), 981-992.

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Transcriptome Analysis of lncRNA and miRNA

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Total RNA was extracted with RNeasy kit (Qiagen, Madison, WI, USA) according to the manufacturer's protocol. Expression of genomic lncRNA was detected using the Affymetrix GeneChip Human Transcriptome Array (HTA2.0; Affymetrix, Santa Clara, CA, USA). Expression of genomic miRNA was detected using the GeneChip® microRNA4.0 array (Affymetrix). The microarray experiments were performed by the Gminix Corporation (Shanghai, China).

Yuan S., Xiang Y., Wang G., Zhou M., Meng G., Liu Q., Hu Z., Li C., Xie W., Wu N., Wu L., Cai T., Ma X., Zhang Y., Yu Z., Bai L, & Li Y. (2019). Hypoxia‐sensitive LINC01436 is regulated by E2F6 and acts as an oncogene by targeting miR‐30a‐3p in non‐small cell lung cancer. Molecular Oncology, 13(4), 840-856.

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Microarray Analysis of miRNA Expression

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A GeneChip microRNA 4.0 array (Thermo Fisher Scientific) containing probes representing 2588 miRNAs (miRBase release 21; www.mirbase.org accessed on 24 August 2018) was performed according to the protocol described by Felix F. Tainara [55 (link)]. The data analysis was executed using Expression Console software v.4.0.2 (Affymetrix, Santa Clara, CA, USA) for data summarization, normalization, and quality control. To identify significantly deregulated miRNAs, the following parameters were used in each tumor compared to normal tissues: fold change (FC) ≥ 2, p-value < 0.0001 and a false discovery rate (FDR) < 0.05 were used to compare each tumor to normal tissues.

Álvarez-Hilario L.G., Salmerón-Bárcenas E.G., Ávila-López P.A., Hernández-Montes G., Aréchaga-Ocampo E., Herrera-Goepfert R., Albores-Saavedra J., Manzano-Robleda M.D., Saldívar-Cerón H.I., Martínez-Frías S.P., Thompson-Bonilla M.D., Vargas M, & Hernández-Rivas R. (2023). Circulating miRNAs as Noninvasive Biomarkers for PDAC Diagnosis and Prognosis in Mexico. International Journal of Molecular Sciences, 24(20), 15193.

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Microarray-based Profiling of Pressure and Shear-Induced miRNAs

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miRs were extracted using a Purelink™ microRNA Isolation Kit (ThermoFisher Scientific, Waltham, MA). Ten microliters of individual samples were mixed into a pooled sample in each group. miR profiling of three pooled samples was outsourced to Filgen, Inc. (Nagoya, Japan). In brief, 336 ng of miR was biotinized using a FlashTag™ Biotin HSR RNA Labeling Kit (ThermoFisher Scientific) and then reacted with a GeneChip® microRNA 4.0 Array (ThermoFisher Scientific), which included probes for 490 rat precursor miRs (pre‐miRs) and 728 mature miRs, at 48°C for 18 hours. After washing, the array was scanned using a GeneChip Scanner 3000 7G (ThermoFisher Scientific).
The results were qualitatively and quantitatively evaluated. In the qualitative evaluation, miRs that were detected only in the pressure or shear groups were chosen (pressure‐ or shear‐specific miRs). In the quantitative evaluation, the miRs whose expression levels were more than two times higher or less than 0.5 times lower in the pressure or shear groups compared with the control group were selected. miRs with no commercially available specific primer and probe set for real‐time RT‐PCR were excluded.

Hsu W., Minematsu T., Nakagami G., Koudounas S., Tomida S., Nakai A., Kunimitsu M., Nitta S, & Sanada H. (2021). Identification of microRNAs responsive to shear loading in rat skin. International Wound Journal, 19(2), 351-361.

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Microarray Analysis of miRNA in AD

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1000 ng of RNA was labeled with Affymetrix FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA, USA) per the manufacturer’s instructions. Labeled RNA was hybridized to GeneChip microRNA 4.0 arrays (Affymetrix, Santa Clara, CA, USA). Chips were run using a Fluidics Station 450 Protocol (Affymetrix, Santa Clara, CA, USA). The results were exported to Partek Genomics Suite (version 6.6, Partek, St. Louis, MO, USA) for analysis. For initial comparison, the entire dataset was normalized using RMA-DABG in Expression Console, and data were exported to Partek Genomics Suite. Using this data, the mean probe intensities for each of the 2578 mature miRNA probes in the CSF and serum were plotted for all paired samples (n = 21). Next, the raw data from Expression console was exported, and normalization was carried out within each biofluid using RMA-DABG. Mature human miRNAs were analyzed and compared between disease groups in Partek Genomics Suite. ANOVA was used to compare miRNAs between patients with AD and controls. Serum and CSF were analyzed separately [49 (link)].

Batabyal R.A., Bansal A., Cechinel L.R., Authelet K., Goldberg M., Nadler E., Keene C.D., Jayadev S., Domoto-Reilly K., Li G., Peskind E., Hashimoto-Torii K., Buchwald D, & Freishtat R.J. (2023). Adipocyte-Derived Small Extracellular Vesicles from Patients with Alzheimer Disease Carry miRNAs Predicted to Target the CREB Signaling Pathway in Neurons. International Journal of Molecular Sciences, 24(18), 14024.

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Profiling Adipocyte-Derived Extracellular Vesicles

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Adipocyte-derived EVs were isolated using the commercially available EoxQuick Precipitation Solution (System Biosciences, Mountain View, CA) from the serum of an all-female subset, chosen to be phenotypically representative of the larger cohort, as previously described [15 (link)]. Total RNA was extracted from adipocyte-derived EVs using the commercially available SeraMir Exosome RNA Amplification Kit (System Biosciences, Mountain View, CA) according to manufacturer instructions. RNA was labeled with Affymetrix^® FlashTag™ Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA) according to standard procedures. Labeled RNA was hybridized to Affymetrix GeneChip microRNA 4.0 arrays and run using a Fluidics Station 450 Protocol (FS450_002) (Affymetrix, Santa Clara, CA). MicroRNAs and ProbeIDs used for statistical analysis are provided in Additional file 2: Table S1 (Accession #: GSE125494).

Barberio M.D., Kasselman L.J., Playford M.P., Epstein S.B., Renna H.A., Goldberg M., DeLeon J., Voloshyna I., Barlev A., Salama M., Ferrante S.C., Nadler E.P., Mehta N., Reiss A.B, & Freishtat R.J. (2019). Cholesterol efflux alterations in adolescent obesity: role of adipose-derived extracellular vesical microRNAs. Journal of Translational Medicine, 17, 232.

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Microarray Analysis of miRNA Expression

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We labeled 1000 ng RNA with Affymetrix® FlashTag™ Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA) according to standard procedures. Labeled RNA was hybridized to Affymetrix GeneChip microRNA 4.0 arrays and run using a Fluidics Station 450 Protocol (FS450_002) (Affymetrix, Santa Clara, CA). Data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession GSE74083 (http://www.ncbi.nlm.nih.gov/geo/).
Resulting data were analyzed in Expression Console using RMA+DMBG (Affymetrix) and then exported to Partek Genomics Suite for analyses. Only mature human microRNAs were retained for statistical comparison between groups. Repeated measures ANCOVA (time; age covariate) was used to compare microRNA expression from pre- to post-surgery, using p≤0.05 as a filter. Rather than use a False Discovery Rate on the initial microRNA dataset, we carried all microRNAs that met the unadjusted p<0.05 cutoff into biological pathway analysis and used more stringent cutoffs during the pathway identification process (as described below). The relationship between insulin resistance (HOMA) and microRNA changes from baseline to one-year post-surgery was also assessed using Pearson correlation coefficients.

Hubal M.J., Nadler E.P., Ferrante S.C., Barberio M.D., Suh J.H., Wang J., Dohm G.L., Pories W.J., Mietus-Snyder M, & Freishtat R.J. (2016). CIRCULATING ADIPOCYTE-DERIVED EXOSOMAL MICRORNAs ASSOCIATED WITH DECREASED INSULIN RESISTANCE AFTER GASTRIC BYPASS. Obesity (Silver Spring, Md.), 25(1), 102-110.

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Nasal Airway miRNA Extraction and Profiling

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Total RNA from the nasal airway specimens was isolated using a Norgen RNA/DNA Purification Kit (Norgen Biotek, Thorold, ON, Canada) and amplified using a Seramir Exosome RNA Amplification Kit (System Biosciences, Palo Alto, CA). MicroRNA quality was determined by Nanodrop1000 (Thermo Scientific, Wilmington, DE) with absorbance ratios for UV 260/280 ≥2.0 and 260/230 between 1.8 and 2.2. Samples meeting quality control criteria were hybridized to Affymetrix GeneChip microRNA 4.0 arrays (Affymetrix, Santa Clara, CA). Resulting data were analyzed in Expression Console using RMA+DMBG (Affymetrix) then exported to Partek Genomics Suite (Partek Inc., St. Louis, MO) for the analyses.

Hasegawa K., Pérez-Losada M., Hoptay C.E., Epstein S., Mansbach J.M., Teach S.J., Piedra P.A., Camargo CA J.r, & Freishtat R.J. (2018). RSV bronchiolitis versus rhinovirus: Difference in nasal airway microRNA profiles and NFκB signaling. Pediatric research, 83(3), 606-614.

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