Low input quick amp labelling kit

Gene expression data were generated from high-quality RNA samples on an Agilent microarray platform (Agilent Technologies). RNA was labelled with a Low Input Quick Amp Labelling Kit (Agilent Technologies) according to the manufacturer’s instructions. Quantity and labelling efficiency were verified before hybridisation to whole-genome 8 × 60 k mouse expression arrays (Agilent design ID 028005) and scanned at 5 μm using an Agilent scanner. Image analysis and data extraction were performed with the Agilent’s Feature Extraction software (version 11.5) to generate the raw expression data.

We used the same total RNA that was used in the miRNA microarray. Total RNA was labelled with the Low Input Quick Amp Labelling Kit (Agilent Technologies, Inc.) and hybridized using the Gene Expression Hybridization Kit (Agilent Technologies, Inc.). The Sureprint G3 Human Gene Expression V3 array (26,083 Entrez Genes, 30,606 lncRNAs, Agilent Technologies, Inc.) was used according to the manufacturer's protocol. The chips were scanned using a SureScan MicroArray Scanner (G2600I, Agilent Technologies, Inc.), and the signal values were analyzed using Feature Extraction software 12.0.3.1 (Agilent Technologies, Inc.).

100 ng total RNA was converted to cDNA, amplified and labelled with Cy-3 using LowInput QuickAmp Labelling Kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. The global transcription profiles were evaluated using the SurePrint G3 Human GE 8x60K microarrays (Agilent Technologies, Santa Clara, CA, USA). The mRNA microarray expression profiles from three cell lines with three replicates for two cell lines, four replicates for one cell line, three fresh frozen tumour samples and two corresponding normal tissue samples were background corrected and quantile normalised. Removal of control probes, un-annotated genes and low expressed probes was done by calculating the 95% percentile of the negative control probes on each array. Probes that were at least 10% brighter than the negative controls on at least 50% of the arrays were kept. Further, 6810 differentially expressed mRNAs were removed by carrying out a moderated t-test for cell line expression versus tumour samples, since these genes may result from contaminating normal and stromal cells in tumour tissues or proliferation associated mRNAs. The unsupervised hierarchical clustering was carried out using Pearson and Spearman's rank correlation coefficient using 10 948 expressed mRNAs. The data are available through Gene Expression Ominbus (GSE58561).

Total RNAs (50 ng) from MCF7-luc derivatives (MCF7-luc anti-NC, MCF7-luc anti-miR-27b, MCF7-luc miR-27b o.e. and MCF7-ENPP1-MF cells) were labelled and amplified using the Low Input Quick Amp labelling kit (Agilent Technologies). The Cy3-labelled RNAs were resuspended in 40 μl of hybridization solution (Agilent Technologies), applied to a SurePrint G3 Human GE 8 × 60 K array (Agilent Technologies) and covered with a Gasket 8-plex slide (Agilent Technologies). The slides were hybridized for 17 h at 65 °C, washed with Gene Expression Wash Buffer 1 (Agilent Technologies) for 1 min at room temperature, washed with Gene Expression Wash Buffer 2 (Agilent Technologies) for 1 min at 37 °C and then air-dried. The arrays from three independent experiments were analysed using microarray scanner (Agilent Technologies). Gene expression levels were calculated using Feature Extraction version 10.7.3.1 (Agilent Technologies). The normalized and log-transformed intensity values were then analysed using GeneSpring GX 7.3.1 (Agilent Technologies).

Total RNA obtained from PBMC of CLL patients (n = 21) and CD19+ sorted cells pooled from 10 healthy individuals was amplified and simultaneously labelled with Cy3-CTP using low input quick amp labelling kit (Agilent Technologies, Santa Clara, CA, USA). The labelled product was finally hybridized to SurePrint G3 Human Gene Expression 8x60K microarray slide as per manufacturer’s recommendation (Agilent Technologies, Santa Clara, CA, USA). The slides were washed and scanned on the Agilent DNA microarray scanner D and the data was extracted with Feature Extraction® software FE version 11.5 (Agilent Technologies, Santa Clara, CA, USA). These samples included seven CLL samples profiled for DNA methylation status.

Total RNA was extracted from collected samples, using a Nucleo Spin RNA plant kit (Macherey-Nagel, Düren, Germany), and then converted to cRNA and labelled using a Low Input Quick Amp Labelling Kit (Agilent Technologies, Santa Clara, CA, USA) and fluorophore Cyanine 3-cytidine triphosphate (cy3-CTP). The samples were cleaned using an RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany). The evaluation of RNA quantity and quality was conducted using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and 1.5% agarose gel electrophoresis.