Sureprint g3 human gene expression v3 8 60k microarray

Manufactured by Agilent Technologies

Sourced in United States, China

The SurePrint G3 Human Gene Expression v3 8 × 60K Microarray is a high-density microarray designed for comprehensive gene expression profiling. It contains approximately 60,000 probes representing human genes and transcripts. The microarray is divided into 8 identical sub-arrays, allowing for the simultaneous analysis of multiple samples.

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13 protocols using sureprint g3 human gene expression v3 8 60k microarray

Transcriptome Analysis via Microarray

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Total RNA was prepared using an RNeasy mini kit (Qiagen). Purified and labeled RNA was hybridized to an Agilent SurePrint G3 Human Gene Expression v3 8 × 60K Microarray (Design ID: 072363, Agilent, Inc., Santa Clara, CA, USA) in accordance with the manufacturer’s instructions. Microarray results were extracted using Agilent Feature Extraction software v11.0 (Agilent Technologies).

Araki R., Hoki Y., Suga T., Obara C., Sunayama M., Imadome K., Fujita M., Kamimura S., Nakamura M., Wakayama S., Nagy A., Wakayama T, & Abe M. (2020). Genetic aberrations in iPSCs are introduced by a transient G1/S cell cycle checkpoint deficiency. Nature Communications, 11, 197.

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Differential Gene Expression in MDA-MB-231 Cells

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Total RNA was extracted from MDA-MB-231 cells transfected with control siRNA or si-circWWC3. Agilent SurePrint G3 Human Gene Expression v3 8 × 60 K Microarray was used to identify differentially expressed genes. The nucleic acid preparation and microarray hybridization process were carried out based on Agilent’s protocols.

Zheng Y., Ren S., Zhang Y., Liu S., Meng L., Liu F., Gu L., Ai N, & Sang M. (2022). Circular RNA circWWC3 augments breast cancer progression through promoting M2 macrophage polarization and tumor immune escape via regulating the expression and secretion of IL-4. Cancer Cell International, 22, 264.

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Microarray Analysis of Gene Expression

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RNA concentration and purity were evaluated using a NanoDrop 2000c UV-Vis spectrophotometer (Thermo Scientific, United States). The RNA integrity number (RIN) was measured for each sample using Agilent 2100 Expert software. Samples with a RIN value of 6 and higher were further processed (Schroeder et al., 2006 (link)). The SurePrint G3 Human Gene Expression v3 8 × 60K Microarray (Agilent, United States) was utilized in this study. The arrays were processed according to the One-Color Microarray Based Gene Expression Analysis protocol v. 6.9.1. The slides were scanned using a SureScan Dx Microarray Scanner (Agilent, United States). The images obtained after scanning were analyzed using Agilent Feature Extraction software v. 12.0.3.1. The analysis included quality control metrics (filtering of outlier spots, background subtraction from features, and dye normalization) and report. An image and a detailed description of the quality control metrics are in Supplementary Excel File_1-QC Metrics.
The principal component analysis (PCA) was conducted on microarray data (submitted to GEO NCBI resources) using filtered flags [detected, not detected] (Figure 1).

Dmitrzak-Weglarz M., Banach E., Bilska K., Narozna B., Szczepankiewicz A., Reszka E., Jablonska E., Kapelski P., Skibinska M, & Pawlak J. (2021). Molecular Regulation of the Melatonin Biosynthesis Pathway in Unipolar and Bipolar Depression. Frontiers in Pharmacology, 12, 666541.

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LEDGF/p75 Knockdown in 786-O Cells

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786-O cells were selected for LEDGF/p75 knockdown treatment, and three biological replicates were prepared for gene microarray detection. Agilent SurePrint G3 Human Gene Expression v3 8×60K Microarray (DesignID:072363) chip experiments and data analysis of 6 samples were performed at Shanghai Ouyi Biomedical Technology Co., Ltd. China.
Feature Extraction software version 10.7.1.1 (Agilent Technologies) was used to process original images and extract original data. The original data were then standardized. Differential genes were screened according to fold change > 1.5 and P value < 0.05. Then, GO and KEGG enrichment analyses of differentially expressed genes were performed to determine biological functions and pathways.

Zhang Y., Guo W., Feng Y., Yang L., Lin H., Zhou P., Zhao K., Jiang L., Yao B, & Feng N. (2023). Identification of the H3K36me3 reader LEDGF/p75 in the pancancer landscape and functional exploration in clear cell renal cell carcinoma. Computational and Structural Biotechnology Journal, 21, 4134-4148.

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Profiling miRNA and mRNA Abundance

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The abundance level of miRNAs was performed using the SurePrint™ 8 × 60 K Human v21 miRNA microarrays (Agilent Technologies), according to the manufacturer’s instructions with slight modification. Briefly, 125 ng RNA was labeled, hybridized to the miRNA microarray chip, washed, and the images were acquired using an Agilent DNA microarray scanner (Agilent Technologies). Similarly, the abundance level of mRNAs was performed using the same samples by hybridization onto SurePrint G3 Human Gene Expression v3 8 × 60K microarrays, containing 50,599 biological features (Agilent Technologies), according to the manufacturer’s instructions with slight modification. Briefly, 125 ng total RNA was reverse transcribed, amplified, labeled with cyanine-3 (Cy3), and subsequently hybridized to the mRNA microarray chip. Arrays were washed, and images were acquired using an Agilent DNA microarray scanner (Agilent Technologies). Finally, the Feature Extraction Software (Agilent Technologies) was used to extract miRNA and mRNA expression data.

Abu-Halima M., Wagner V., Becker L.S., Ayesh B.M., Abd El-Rahman M., Fischer U., Meese E, & Abdul-Khaliq H. (2021). Integrated microRNA and mRNA Expression Profiling Identifies Novel Targets and Networks Associated with Ebstein’s Anomaly. Cells, 10(5), 1066.

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Microarray Analysis of Epithelial and Stromal Gene Expression

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Gene expression profiles of epithelial and stromal samples of KC and normal samples were measured using SurePrint G3 Human Gene expression v3 8×60K microarrays and the Low Input Quick Amp Labeling Kit (Agilent Technologies). Total RNA (100 ng) of each sample was used for complementary DNA (cDNA) synthesis using a T7 primer. The cDNA was transcribed into cRNA using a Cy3-labeled pCp and T7 polymerase master mix. Following determination of RNA concentration, samples were hybridized to microarrays and scanned using an Agilent microarray scanner with a resolution of 3 µm, followed by analysis using Agilent Feature Extraction software (version 10.10.1.1).

Stachon T., Nastaranpour M., Seitz B., Meese E., Latta L., Taneri S., Ardjomand N., Szentmáry N, & Ludwig N. (2022). Altered Regulation of mRNA and miRNA Expression in Epithelial and Stromal Tissue of Keratoconus Corneas. Investigative Ophthalmology & Visual Science, 63(8), 7.

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RNA Extraction and Microarray Analysis Protocol

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RNA was extracted from PAXgene tubes using the PAXgene Blood miRNA Kit (Qiagen, Hilden, Germany). 25 ng RNA was amplified and labelled with Cy3 using the Low Input Quick Amp Labeling Kit, one-color (Agilent, CA, USA), and the labelled cRNA was purified using the Qiagen RNeasy Mini Kit. Amplification and labelling efficiency were controlled on a NanoDrop ND-1000 spectrophotometer (Thermo Fisher, MA, USA). cRNA quality was assessed by measuring the RNA absorbance ratio 260 nm/280 nm. The specific activity of labeled cRNA (pmol Cy3 per ug cRNA) was defined to be 6 or above. 19

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L of cRNA per sample was fragmented and applied to Agilent SurePrint G3 Human Gene Expression v3 8 × 60 k Microarrays. After hybridization for 17 h at 65 °C the microarray slides were washed, scanned with the Agilent Microarray Scanner and the data extracted using Agilent Feature Extraction Software.

Prebensen C., Lefol Y., Myhre P.L., Lüders T., Jonassen C., Blomfeldt A., Omland T., Nilsen H, & Berdal J.E. (2023). Longitudinal whole blood transcriptomic analysis characterizes neutrophil activation and interferon signaling in moderate and severe COVID-19. Scientific Reports, 13, 10368.

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mRNA Expression Profiling in Heart Diseases

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Similarly, the expression level of mRNAs was performed using the same samples that were used for the miRNA microarray analysis by hybridization onto SurePrint G3 Human Gene Expression v3 8 × 60K microarrays, containing 50,599 biological features (Agilent Technologies), according to the manufacturer’s instructions with slight modification. mRNA expression profiling was carried out on TGA-RV (n = 16), TGA-LV (n = 16), and controls (n = 16). Briefly, 125 ng total RNA was reverse transcribed, amplified, labeled with cyanine-3 (Cy3), and subsequently hybridized to the mRNA microarray chip. Arrays were washed, and images were acquired using an Agilent DNA microarray scanner (Agilent Technologies). Finally, generated data were imported into R statistical environment software (version R-4.1.2) for further statistical analysis.

Abu-Halima M., Wagner V., Rishik S., Raedle-Hurst T., Meese E, & Abdul-Khaliq H. (2023). Expression profiling analysis reveals key microRNA–mRNA interactions in patients with transposition of the great arteries and systemic left and right ventricles. Frontiers in Cardiovascular Medicine, 9, 1056427.

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Gene Expression Microarray Analysis Protocol

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cRNA amplification, labeling, hybridization, and analysis were performed at Takara Bio using the SurePrint G3 Human gene expression 8×60K v3 microarray (Agilent Technologies). The GEO accession number for the microarray analysis is GEO: GSE131550.

Kawai K., Negoro R., Ichikawa M., Yamashita T., Deguchi S., Harada K., Hirata K., Takayama K, & Mizuguchi H. (2019). Establishment of SLC15A1/PEPT1-Knockout Human-Induced Pluripotent Stem Cell Line for Intestinal Drug Absorption Studies. Molecular Therapy. Methods & Clinical Development, 17, 49-57.

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Transcriptome Analysis of NIH 3T3 Cells

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The transcriptomes of the paired sCA-siCON- and sCA-siTIMP1-transfected NIH 3T3 cells were analyzed using an Agilent Technologies-based microarray platform with 8 × 60K probes. Total RNA from cells was translated into cRNA and labeled with Cy3 using the Agilent Quick Amp labeling kit (Agilent). The labeled cRNA was then purified using an RNeasy mini kit (QIAGEN). An Agilent ND-1000 was used to determine the concentration and specific activity of the labeled cRNA. Labeled cRNA from the paired control and treated samples was hybridized to the SurePrint G3 human gene expression 8 × 60K v3 microarray (Agilent). The signals were scanned using SureScan G4900DA (Agilent).

Aoki M., Matsumoto N.M., Dohi T., Kuwahawa H., Akaishi S., Okubo Y., Ogawa R., Yamamoto H, & Takabe K. (2020). Direct Delivery of Apatite Nanoparticle-Encapsulated siRNA Targeting TIMP-1 for Intractable Abnormal Scars. Molecular Therapy. Nucleic Acids, 22, 50-61.

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