Total RNA was isolated from 1e7 freshly isolated PBMCs using miRNeasy kit (QIAGEN) according to the manufacturer’s instructions and eluted in 40 µl of elution buffer with the addition of 1 µl of RNase inhibitor (Life Sciences, Carlsbad, CA, USA). RNA quality was assessed by Agilent Bioanalyzer and quantified by NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). 300 ng of total RNA were amplified using Ambion WT expression kit (Life Sciences) according to the manufacturer’s instructions. Fragmented single-stranded sense cDNA were terminally labeled and hybridized to Human 1.0 ST GeneChip arrays (Affymetrix, Santa Clara, CA, USA) and stained on a GeneChip Fluidics Station 450 (Affymetrix), according to the respective manufacturers’ instructions. Arrays were scanned on a GeneChip Scanner 3000 7G (Affymetrix).
Genechip human 1.0 st arrays
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip Human 1.0 ST Arrays is a high-density oligonucleotide array designed for gene expression analysis. The array targets over 28,000 well-annotated genes, providing comprehensive coverage of the human transcriptome.
Automatically generated - may contain errors
Lab products found in correlation
Genechip scanner 3000 7g, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Mirneasy kit, by Qiagen (1 mentions)
Rna nano chip, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Quick rna microprep kit, by Zymo Research (1 mentions)
Power tools software, by Thermo Fisher Scientific (1 mentions)
Genechip scanner gcs3000, by Thermo Fisher Scientific (1 mentions)
Ambion wt expression kit for affymetrix genechip whole transcript expression arrays, by Thermo Fisher Scientific (1 mentions)
6 protocols using genechip human 1.0 st arrays
1
Total RNA Isolation and Microarray Analysis
2
Microarray Analysis of Extracted mRNA
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After treatment, total mRNA from the cells was extracted using the Quick-RNA Microprep kit (Zymo Research, Freiburg, Germany), according to the manufacturer's instructions. Quality and concentration of the mRNA samples were determined using the RNA Nano chip (Agilent Technologies) and analyzed by the Agilent 2100 Bioanalyzer (Agilent Technologies). mRNA extracts were stored at -80°C until further use for array analysis. Microarray experiments were conducted according to the manufacturer's instructions (Central Research Unit-INCLIVA; University of Valencia, Spain). RNA was hybridized to Human 1.0 ST GeneChip Arrays (Affymetrix). The array includes >750,000 unique 25-mer oligonucleotide transcripts, thus constituting >28,000 well-annotated genes. Analyses were performed by using one patient sample per GeneChip. Files were captured using an Affymetrix GeneChip Scanner 3000 7G. To verify if microarray results were reliable and sample quality was good, RNA samples from four of the 12 conditions were randomly selected to be replicated as a technical control.
3
Affymetrix GeneChip Analysis of Liver Samples
4
Microarray Hybridization and Analysis
5
Gene Expression Profiling with GeneChip Arrays
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This procedure requires the use of the GeneChip® Hybridization, Wash and Stain Kit (catalog no. 900720, Affymetrix) and the GeneChip human 1.0 ST Arrays (catalog no. 901085, Affymetrix). Hybridization cocktail was prepared by mixing the following materials: 25 microliter of fragmented, biotin-labeled and amplified cDNA, 1.9 microliter control oligonucleotide B2 (3 nM), 5.5 microliter of 20X Eukaryotic hybridization controls (bioB, bioC, bioD, cre), 55 microliter 2X Hybridization Buffer, 11 microliter 100% DMSO, 11.6 microliter water. The procedure was performed according to manufacturer instructions.
6
Transcriptome Analysis of Placental Cells in GDM
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Total RNA from AEC (n = 8) and VEC (n = 8) isolated from eight placentas from normal pregnancies and dAEC (n = 11) and dVEC (n = 10) isolated from 14 placentas from pregnancies complicated by GDM was labelled using Ambion WT Expression Kit for Affymetrix GeneChip Whole transcript (WT) Expression Arrays (Life Technologies, Carlsbad, CA). The cRNA was hybridised to GeneChip Human 1.0 ST arrays according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA). Washing and staining (GeneChip HT Hybridization, Wash and Stain Kit; Affymetrix) was performed with Affymetrix GeneChip fluidics station 450. Arrays were scanned using Affymetrix GeneChip scanner GCS3000. Labelling and hybridisation controls were evaluated with Affymetrix Expression Console EC 1.1. Data were analysed with RMA (robust multi-chip average), including background correction, quantile normalisation, log2 transformation and median polish summarisation using Genomic Suite v6.5 (Partek, St Louis, MO, USA) [24 (link)]. Statistical analysis used one-way ANOVA with fetal sex and mother as random factors. The p values were adjusted for multiple testing using the Benjamini–Hochberg method (R/Bioconductor package ‘multtest’; https://www.bioconductor.org/packages/release/bioc/html/multtest.html ) [25 (link), 26 ].
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