G2505c microarray scanner system

Manufactured by Agilent Technologies

The G2505C Microarray Scanner System is a high-resolution, two-color microarray scanner designed for the analysis of DNA, protein, and other biological samples. The system features a compact design, flexible scanning options, and automated data extraction capabilities.

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8 protocols using g2505c microarray scanner system

Genome-wide Microarray Analysis of Cell Differentiation

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Total RNA was extracted from frozen cell samples using ISOGEN (NIPPON GENE) according to the manufacturer’s instructions. Samples were analyzed using an Agilent SurePrint G3 Human GE 8 × 60K Microarray Kit (G4851A) and a one-color Low Input Quick Amp Labeling Kit (Agilent). Arrays were scanned using a G2505C Microarray Scanner System (Agilent). Raw microarray data were submitted to the Gene Expression Omnibus (GEO) microarray data archive (http://www.ncbi.nlm.nih.gov/geo/) at NCBI (accession number GSE55428).
Data were analyzed using Gene-Spring GX12.0 software (Agilent) after applying two normalization procedures. First, signal intensities of less than 1 were set to 1, and then each chip was normalized to the 75th percentile of all measurements from that chip. Baseline transformation of these data was not performed. Genes with a flag value of “detected” or “not detected” in at least one sample were analyzed. Differentially expressed genes were selected if the average values of each sample were altered by at least 2.0-fold. Early differentiation marker genes for the heat map were selected as previously described [21 (link)]. GO terms enriched with a p-value cut-off of 0.01 were extracted.

Onuma Y., Higuchi K., Aiki Y., Shu Y., Asada M., Asashima M., Suzuki M., Imamura T, & Ito Y. (2015). A Stable Chimeric Fibroblast Growth Factor (FGF) Can Successfully Replace Basic FGF in Human Pluripotent Stem Cell Culture. PLoS ONE, 10(4), e0118931.

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Gene Expression Profiling of Dental Pulp Cells

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Total RNA was prepared using the RNeasy Midi Kit (Qiagen). RNA (250 ng) was converted to cDNA, amplified, and labeled with Cy3 using the Quick Amp Labeling Kit (Agilent). Labeled DNA was hybridized to a Whole Human Genome Microarray Kit, 4 × 44 K (Agilent) according to the manufacturer's protocol. After hybridization, the arrays were washed consecutively using a Gene Expression Wash Pack (Agilent) and scanned using the G2505C Microarray Scanner System (Agilent). The data were normalized and analyzed by using GeneSpringGX11 (Agilent). The level of gene expression was determined as the average difference, and we selected genes with at least a five-fold difference in expression between DPCs from immature teeth and those from mature teeth. The microarray data was submitted to the NCBI GEO database under the accession number (GSE52853).

Tamaoki N., Takahashi K., Aoki H., Iida K., Kawaguchi T., Hatakeyama D., Inden M., Chosa N., Ishisaki A., Kunisada T., Shibata T., Goshima N., Yamanaka S, & Tezuka K.I. (2014). The homeobox gene DLX4 promotes generation of human induced pluripotent stem cells. Scientific Reports, 4, 7283.

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Circular RNA Enrichment and Microarray Analysis

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RNA samples (n = 4 per group) were prepared with Eastep® Super RNA Extraction Kit (Promega (Beijing) Biotech Co., Ltd.), followed by RNase R (Epicentre) digestion to eliminate linear RNAs and enrich circRNAs in the RNA samples. The purity and yields of RNA samples were evaluated using a NanoDrop spectrophotometer (ND‐1000; NanoDrop, Inc). Then, the enriched circRNAs were amplified and transcribed into fluorescence‐labelled coding RNAs using a random priming approach (Super RNA Labeling Kit; Arraystar). The labelled coding RNAs were then hybridized onto the Human circRNAs Array v2 (8 × 15 K, Arraystar). After washing, a G2505C Microarray Scanner System (Agilent Technologies, Inc) was used to perform the microarray analysis.

Liu X., Du Z., Yi X., Sheng T., Yuan J, & Jia J. (2021). Circular RNA circANAPC2 mediates the impairment of endochondral ossification by miR‐874‐3p/SMAD3 signalling pathway in idiopathic short stature. Journal of Cellular and Molecular Medicine, 25(7), 3408-3426.

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Microarray Analysis of Spleen Transcriptome

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For microarray-based gene-expression analysis, we pooled equal shares of seven individual RNA samples isolated from the spleen in six separate specimens according to treatment and tank (A1, B1, C1; A2, B2, C2). These six RNA pools were converted to Cy3-labelled cRNA and hybridised with 8 × 60 K Agilent-049158 Salmon Oligo Microarrays (Agilent Technologies; GEO platform: GPL21057) following the Agilent 60-mer oligo microarray processing protocol, as described in our previous paper³⁶. The fluorescence signals of the hybridised Agilent microarrays were scanned with a G2505C Microarray Scanner System (Agilent Technologies) at a resolution of 3 µm.
For all hybridisations, two technical replicates were included representing exactly the same samples but applied to independent arrays. The reliability of the microarray-predicted data has previously been proven in various quantitative PCR studies of our group^{9 ,23 (link),69 (link),82 (link),83 (link)}.

Rebl A., Korytář T., Borchel A., Bochert R., Strzelczyk J.E., Goldammer T, & Verleih M. (2020). The synergistic interaction of thermal stress coupled with overstocking strongly modulates the transcriptomic activity and immune capacity of rainbow trout (Oncorhynchus mykiss). Scientific Reports, 10, 14913.

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Wheat Kinase Expression Analysis

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For each sample total RNA was extracted from the sheaths of 10 seedlings from sharp eyespot-resistant wheat lines (CI12633 and Shanhongmai) or the susceptible wheat cv. Wenmai 6 at 4 or 21 dpi with R. cerealis. After purification, the RNA was used for the preparation of Cy5- and Cy3-labelled complementary RNA. These probes of Cy3-labelled CI12633/Shanhongmai RNA and Cy5-labelled Wenmai 6 RNA were hybridized with Agilent Wheat Gene Expression Microarray containing 43 803 probe sets according to the manufacturer’s protocols. The hybridization signals were scanned with an Agilent G2505C Microarray Scanner System, and microarray data were extracted using Feature Extraction Software (v.10.7.1.1) available from Agilent by using the default variables. Data files were loaded into GeneSpring GX 11.5 (Agilent Technologies). The signal values between both experimental groups (CI12633 or Shanhongmai probe vs Wenmai 6 probe) were assessed for each gene based on the normalized probe signals. Differentially expressed kinase genes between CI12633 or Shanhongmai and Yangmai 12 were filtered out by a fold change threshold ≥2.0.

Zhu X., Yang K., Wei X., Zhang Q., Rong W., Du L., Ye X., Qi L, & Zhang Z. (2015). The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis. Journal of Experimental Botany, 66(21), 6591-6603.

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Differential miRNA Expression Profiling in Mortality

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To screen out the different miRNA expression profiles by high-throughput microarray technique, we briefly divided the cohort by vita status, specifically, death vs. alive groups, and 4 cases from each group were chosen. The total RNA of 100 ng was labeled with Cy3 fluorescent label after dephosphorylation and denaturation. Purified samples were hybridized with Agilent Human miRNA microarray (Release 21.0, 8*60 K). The arrays were washed twice and scanned by the Agilent G2505C Microarray Scanner System. Image analysis was carried out using the Agilent Feature Extraction Software. We used the Agilent GeneSpring GX Software the correct the qualified signals to background and normalized them by quantile method. For every miRNA probe, fold change (FC) value was calculated (by a formula 2^ [average value of death group – average value of survival group]). If the average value for the death group was greater than that for the alive group, this miRNA expression was labeled with upregulation and vice versa. Criteria for selecting candidate differently expressed miRNA were: (1) FC of miRNA between the two groups ≥2 and p value ≤.05 (2) signals of miRNA probe could be detected in all 8 samples.

Xu X., Liu H., Gross N., Wei D., Qian Y., Li W., Wei P., Li G., Zhang F., Yang Z., Lei D, & Pan X. (2018). Overexpression of miRNA 4451 is Associated With a Poor Survival of Patients With Hypopharyngeal Cancer After Surgery With Postoperative Radiotherapy. Translational Oncology, 11(5), 1244-1250.

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Gene Expression Analysis by Microarray

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Expression analysis by DNA microarray was performed with the Agilent system (G4846A; Whole Mouse Genome Microarray Kit (4x44K) Agilent Technologies, Santa Clara, CA, USA). The probes for the microarray were prepared and labeled by Cy3 according to the manufacturer’s protocol (Agilent Technologies). Arrays were scanned with a G2505C Microarray Scanner System (Agilent Technologies). Data were normalized using the R stats package with the qspline function of the “affy” package in the Bioconductor [22 (link)].

Furuse T., Miyake K., Kohda T., Kaneda H., Hirasawa T., Yamada I., Kushida T., Kashimura M., Kobayashi K., Ishino F., Kubota T, & Wakana S. (2017). Protein-restricted diet during pregnancy after insemination alters behavioral phenotypes of the progeny. Genes & Nutrition, 12, 1.

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Transcriptome Analysis of ATDC5 Cells

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The transcriptome of pressurized and unpressurized ATDC5 cells was analyzed using the SurePrint G3 Mouse Gene Expression v2 8x60K Microarray Kit (G4852B, Agilent) containing just over 56,000 probes (representing 27,122 genes and 4,578 long non-coding RNAs). Two time-course experiments (consisting of control, 1 h HP, 4 h HP and 24 h HP samples) were carried out, producing 8 RNA samples. The quality of the RNA was checked by agarose gel electrophoresis and found to be satisfactory, with two clear bands and no sign of degradation (Fig 1b). The RNA was then reverse-transcribed, labeled with Cy3 and hybridized to one microarray according to the manufacturer’s instructions. After hybridization, fluorescence signals were measured with a G2505C microarray scanner system (Agilent).

Montagne K., Onuma Y., Ito Y., Aiki Y., Furukawa K.S, & Ushida T. (2017). High hydrostatic pressure induces pro-osteoarthritic changes in cartilage precursor cells: A transcriptome analysis. PLoS ONE, 12(8), e0183226.

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