Affymetrix genechip human exon 1.0 st arrays

The 293T cells were incubated in the presence or absence of 10 µM CX-4945 for 12 hours, and total RNAs were purified using the TRIzol reagent. The fragmented and end-labeled single-stranded cDNAs were prepared and hybridized to Affymetrix-GeneChip Human Exon 1.0 ST arrays (Affymetrix). Affymetrix Expression Console Software was used to perform quality assessment. Affymetrix exon array data was analyzed using GeneSpring 12.6 inclusive of GX (Agilent Technologies). Three independent experimental samples were examined.

Affymetrix exon array hybridization was completed by labeling 1 μg of TRIzol-purified total RNA with Affymetrix reagents according to the manufacturer’s instructions. Hybridization cocktails containing 5–5.5 μg cDNA were prepared and hybridized to Affymetrix-GeneChip Human Exon 1.0 ST arrays (Affymetrix). Affymetrix Expression Console Software was used for quality assessment, while exon array data were normalized using quintile normalization and analyzed using FasterDB annotation (https://fasterdb.lyon.unicancer.fr/) [41 (link)]. Background correction and probe selection were performed as described previously [42 (link)]. Statistical analyses were performed using a Student’s t-test on the splicing index (SI) that corresponds to comparison of gene-normalized exon intensity values between two experimental conditions [30 (link),42 (link)]. According to exon-specific RT-PCR (Figures 1C and D), microarray data were considered statistically significant for p < 0.05 and SI ≥ 1.2.

The frozen oral mucosa samples were homogenized with TRIzol^R Reagent (Invitrogen, ThermoFischer Scientific), and total RNA was isolated with the mirVana^TM miRNA Isolation Kit (Ambion/Invitrogen, ThermoFischer Scientific) according to the manufacturer’s protocol. RNA amplification was performed with the Ambion^R WT Expression Kit (Applied Biosystems, ThermoFischer Scientific), according to the manufacturer’s instructions, starting with 100 ng total RNA, on a TP Basic Thermocycler, real time PCR instrument (Biometra, Göttingen, Germany). The quality of the RNA product was evaluated on the NanoDrop spectrophotometer and the 2100 Bioanalyzer with the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, US). We prepared the RNA samples for hybridization to Affymetrix GeneChip Human Exon 1.0 ST Arrays with the Affymetrix GeneChip WT Terminal Labeling and Controls Kit (P/N 901524) (Affymetrix, Santa Clara, CA, US), according to the manufacturer’s instructions. CEL files were generated with Affymetrix GeneChip Command Console Software and deposited at the NCBI Gene Expression Omnibus repository, under number GSE81979. A similar procedure was applied to analyze gene expression in MNCs isolated from blood samples.