Gene expression analysis were performed as previously described (Ravindran Menon, Das et al. 2015 (link)). Briefly, total RNA was labeled using the Ambion WT Expression Kit for Affymetrix GeneChip whole transcript (WT) Expression Arrays® (Life Technologies) and 250 ng of the labeled total RNA was then hybridized to GeneChip Human 1.0 ST arrays according to the manufacturer’s instruction. The Affymetrix GeneChip scanner GCS3000 was used for reading. Genomic Suite v6.5 (Partek Inc) was used for the analyses of the results. All samples passed the quality control check and significantly differentially expressed genes were determined using ANOVA. Genes with p < 0.05 and FC≥ +/− 1.5 were used for further analysis.
Ambion wt expression kit for affymetrix genechip whole transcript expression arrays
Manufactured by Thermo Fisher Scientific
The Ambion WT Expression Kit for Affymetrix GeneChip whole transcript (WT) Expression Arrays is a laboratory equipment product designed for RNA amplification and labeling. The kit enables the preparation of amplified and biotinylated sense-strand cDNA from total RNA for hybridization to Affymetrix GeneChip whole transcript expression arrays.
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2 protocols using ambion wt expression kit for affymetrix genechip whole transcript expression arrays
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Gene expression analysis protocol
2
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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