Flashtag biotin rna labeling kit

Total RNA of DS (n = 3) and diploid controls (n = 3) was purified by mirVana miRNA Isolation Kit (Ambion). The miRNA expression profiles were generated by using the Affymetrix GeneChip miRNA Array v. 4.0 (Affymetrix). Briefly, the flashTag Biotin RNA Labeling Kit (Affymetrix) was used to label of 1 μg of total RNA, followed by the hybridization overnight according to the manufacturer’s instructions. After washed and stained using the Affymetrix GeneChip Hybridization Wash and Stain Kit, the miRNA chips were then scanned with the Affymetrix GeneChip Scanner 3000 (Affymetrix).

Total RNA from paraffin tissues was isolated using the RNeasy FFPE kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The total RNA was quantified using NanoDrop One (Thermofisher Scientific, Inc., Waltham, MA, USA). For miRNA expression analysis, a standard protocol was followed. Briefly, 250 ng of total RNA from each sample was labeled using the FlashTag™ Biotin RNA Labeling kit (Affymetrix^®; Thermo Fisher Scientific, Inc.), and the labeled RNA was hybridized with the GeneChip miRNA 4.0 Array (Affymetrix^®; Thermo Fisher Scientific, Inc.). The miRNA microarray chips were washed twice with 1X PBST (0.02% Tween) buffer and stained with FlashTag™ Biotin HSR (Affymetrix; Thermo Fisher Scientific, Inc.) for 5 min at 35 °C. CEL files were generated after digitizing the image. The fluorescence intensity values in CEL format were pre-processed and normalized by quartiles using Expression Console™ v1.4.1.46 software (Affymetrix; Thermo Fisher Scientific, Inc.). Transcriptomic Analysis Console™ v4.0 software (Affymetrix; Thermo Fisher Scientific, Inc.) was used for expression analysis and heatmap generation, where the CHP files generated after normalization were loaded. Differentially expressed miRNAs were identified by ±1.5-fold change and eBayes p-values of < 0.05 between each sample group.

Total RNA, containing miRNA, was extracted from MDA-MB-231/FOXO3a cells and MDA-MB-231/control cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The miRNA expression profiles were generated by using the Affymetrix GeneChip miRNA Array v. 4.0 (Affymetrix). Briefly, the flashTag Biotin RNA Labeling Kit (Affymetrix) was used to label 1 μg of total RNA, followed by the hybridization overnight according to the manufacturer’s instructions. Images were scanned using the GeneChip Scanner 3000 and image analysis was done with the GeneChip Operating Software.