Fluidics station fs450

10 × 10⁶ MDS-L cells were treated with WFA (10 μM) or DMSO for 6 h or 12 h and total RNA was extracted using the Direct-zol^™ RNA miniprep kit (Zymo Research #R2051). RNA purity was assessed using the Agilent RNA 6000 Nano assay kit (#5067-1511) on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNA integrity number (RIN) was ≥ 9 for all samples. Sense-strand DNA (ss-cDNA) was generated, amplified and biotinylated using the WT Plus Reagent kit (Affymetrix, Santa Clara, CA) from 100 ng total RNA per sample. 30 μg of fragmented biotin-labelled ss-cDNA was hybridized to Affymetrix human gene 2.0 ST arrays at 45°C and 60 rpm for 16 h. The arrays were washed, stained using the Affymetrix fluidics station FS 450 and scanned on the Affymetrix 7G GeneChip Scanner. The raw microarray data files were processed through Oligo [52 (link)] for data extraction and normalization.
Gene expression profiles of MDS-L cells were examined in triplicate using Affymetrix human gene 2.0 ST arrays. Differential expression analyses comparing WFA-treated and control groups were performed by limma [53 (link)]. Significantly up/downregulated genes were determined as fold change > 3 and q-value < 0.05. Gene set enrichment analysis was performed using GSEA software and the Hallmark gene sets in the Molecular Signature Database (MSigDB) [54 (link)].

Total RNA was isolated using TRI Reagent combined with the RNeasy Tissue kit protocol (Qiagen, Valencia, CA) according to the manufacturer's recommendations. To identified differential genes in HCC tumor tissue, the PrimeView™ Human Gene Expression Array (Affymetrix, Santa Clara, CA) was performed in 3 pairs of HCC tumor and adjacent non-tumor tissues according to the manufacture's protocol. A total of 750 ng of labeled complementary RNAs were hybridized to arrays and then imaged using Affymetrix Fluidics Station FS450 and scanned with GeneChip Scanner 3000 7G according to manufacturer's instructions. Raw signals of the arrays were processed using Affymetrix Power Tools. Data quality was assessed based on the positive and negative control probes on each array as well as by inspection of the distributions of probe intensities. Data was normalized using the quantile normalization method. A moderated t-test implemented in the limma library of bioconductor was applied to test differential expression, and a false discovery rate (FDR) adjustment of the p-value was performed to correct for multiple testing. Probes were considered significantly different if the adjusted p-value was less than 0.05 and the fold change difference between groups was at least 2.

Total RNA of 1 × 10⁶ MPC or MSC, each cultured either in growth medium or 6 days in differentiation medium, was isolated by the RNEasy Kit (QIAGEN, Hilden, Germany) according to the manufacturers’ instructions. Sample preparation for microarray hybridization was carried out as described in the NuGEN Ovation PicoSL WTA System V2 and NUGEN Encore Biotin Module manuals (NuGEN Technologies, Inc., San Carlos, CA, USA). Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. The Affymetrix GeneChip Command Console v4.1.3 software controlled fluidics and scan functions. The Affymetrix Service Provider and Core Facility, “KFB - Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany) performed the sample processing.

Total RNA of SMDCs either cultured in growth medium or 6 days in differentiation medium was isolated by RNEasy Kit (QIAGEN, Hilden, Germany) according to the manufacturers’ instruction. Sample preparation for microarray hybridization was carried out as described in the NuGEN Ovation PicoSL WTA System V2 and NUGEN Encore Biotin Module manuals (NuGEN Technologies, Inc, San Carlos, CA, USA).Briefly, 7.5 ng of total RNA was reverse transcribed into double-stranded cDNA in a two-step process, introducing a SPIA tag sequence. Bead purified cDNA was amplified by a SPIA amplification reaction followed by an additional bead purification. 4.5 μg of SPIA cDNA was fragmented, terminally biotin-labeled and hybridized to Affymetrix PrimeView Human Gene Expression arrays for 16 hours at 45°C in a GeneChip hybridization oven 640. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by the Affymetrix GeneChip Command Console v4.1.3 software.
Sample processing was performed at an Affymetrix Service Provider and Core Facility, “KFB—Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany; www.kfb-regensburg.de).

The RNA samples were prepared for microarray hybridization as described in the Affymetrix GeneChip WT PLUS Reagent Kit User Manual (Affymetrix, Inc., Santa Clara, CA, USA). Labeled ss cDNA was hybridized to Affymetrix Clariom S human arrays for 16 h at 45 °C and 60 rpm in a GeneChip hybridization oven 640. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Sample processing was performed at an Affymetrix Service Provider and Core Facility, “KFB—Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany; www.kfb-regensburg.de). Microarray data analysis and statistics were performed as described previously [43 (link)].

Sample preparation for microarray hybridization was carried out as described in the Affymetrix GeneChip WT PLUS Reagent Kit User Manual (Affymetrix, Inc., Santa Clara, CA, USA).
In brief, 200 ng of total RNA was used to generate double-stranded cDNA. Subsequently, synthesized cRNA (15 µg) was purified and reverse transcribed into sense-strand (ss) cDNA, into which unnatural dUTP residues were incorporated. Purified ss cDNA was fragmented using a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1), followed by terminal labeling with biotin. Then, 5.5 µg fragmented and labeled ss cDNA were hybridized to Affymetrix Clariom D human arrays for 16 h at 45 °C in a GeneChip hybridization oven 640. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by Affymetrix GeneChip Command Console v4.1.3 software.
Sample processing was performed at an Affymetrix Service Provider and Core Facility, “KFB—Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany; www.kfb-regensburg.de, accessed on 20 November 2017).