Totalprep rna amplification kit

RNA was isolated from 5 × 10⁵ DCs on day 5 using total RNA isolation RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Yield and quality of RNA samples were evaluated with NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity (RIN score) was analyzed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) or LabChip GX/GX II (Caliper LifeSciences, Hopkinton, MA, USA). A total of 40 samples, corresponding to 10 healthy donors under four experimental conditions were considered for microarray analysis (Figure S1 in Supplementary Material). All RNA samples used for microarrays showed A260/A280 values between 1.8 and 2.2, and RIN scores >7. RNA samples were reverse transcribed, amplified, and labeled using an Illumina^® TotalPrep™ RNA Amplification Kit, and cDNA was hybridized onto Illumina Human HT-12 v4 BeadChips (Illumina, San Diego, CA, USA), covering the whole human genome. Expression data were extracted with GenomeStudio Project Software from Illumina.

In the METABRIC study,^{10 (link)} mRNA was extracted from primary tumours of female patients, and mRNA expression was evaluated using the Illumina TotalPrep RNA Amplification Kit and Illumina Human HT-12 v3 Expression BeadChips (Ambion, Warrington, UK). LVI status of 1565 patients within the METABRIC cohort, which were histologically assessed using haematoxylin and eosin (H&E) stained slides. For the Nottingham subset included in METABRIC (n = 285/1565), LVI status was additionally assessed by immunohistochemistry (IHC) utilising CD31, CD34 and D2-40,^{12 (link)} and the final LVI status was confirmed using a combination of multiple H&E tumour sections and IHC. Considering the different methods of LVI assessment, cases were divided into two groups: (1) the Nottingham cases and (2) the remaining METABRIC cases (n = 1280). Gene transcript expression levels between LVI-positive and LVI-negative cases were compared for each group, as described in the ‘Bioinformatics analysis’ section.

Total RNA was extracted from CD4+ T cells positively selected from monocyte-depleted whole blood within 4 h of blood draw as previously described [6 (link)]. cRNA generated from 250 ng total RNA (Illumina TotalPrep RNA Amplification Kit) was hybridized to the Illumina Human HT12v4 BeadChip (Illumina, San Diego, CA, USA). After quality control using established methods previously outlined [6 (link)], data relating exclusively to the 12 signature genes previously identified [6 (link)] were extracted for detailed analysis. Expression data used for this experiment are available in the Gene Expression Omnibus database (GEO: http://www.ncbi.nlm.nih.gov/geo; accession number GSE80513). Since the HT12v4 BeadChip annotation differed slightly from the WG6v3 array used for our original work, unique Illumina NuId references were used instead to map probes of identical sequence for this purpose.

For whole-genome expression microarray, RNA was isolated using the RNeasy Plus kit (Qiagen). Biotin labeled complementary RNA (cRNA) was generated using the Illumina TotalPrep RNA amplification kit. Total RNA and cRNA quality were assessed by Bioanalyzer (Agilent). Illumina HumanHT-12 version-4 expression beadchips were hybridized with cRNA from two biological replicates per condition and scanned on an Illumina BeadStation 500GX. Scanned images were converted to raw expression values using GenomeStudio v1.8 software (Illumina). Data analysis was carried out using the statistical computing environment, R (v3.0.2), the Bioconductor suite of packages for R, and RStudio (v0.97). Raw data were background subtracted, variance stabilized, and normalized by robust spline normalization using the Lumi package [71 (link)]. Differentially expressed genes were identified by linear modeling and Bayesian statistics using the Limma package [72 ,73 ]. Probes sets that were differentially regulated (≥1.5 fold, FDR ≦ 5%; after controlling for multiple testing using the Benjamini-Hochberg method [74 (link),75 ]) were used for hierarchical clustering and heatmap generation in R. Clusters of co-regulated genes were identified by Pearson correlation using the hclust function of the stats package in R. Data have been deposited on the Gene Expression Omnibus (GEO) database for public access (GSE55751).

Blood was collected retro-orbitally at 10 weeks of age from female NOD.CD45.2 and C57Bl/6 mice and stored in RNAlater solution (Ambion) at −80 °C. RNA was extracted using Ribo-Pure Blood kit (Ambion), the RNA integrity and concentration were measured using 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). RNA was depleted of alpha and beta globin mRNA using GLOBINclear (Ambion) before amplification with TotalPrep RNA Amplification Kit (Illumina). Resulting cRNA was hybridized to MouseRef-8 v2.0 Expression BeadChip (Illumina).

Illumina Human HT-12v3 BeadChip arrays were performed as described (29 (link)). RNA was isolated from three biological replicates of adipogenic-BMSCs at day 4 and day 13 as described (28 (link)). RNA concentration and size distribution profiles were assessed on a 2100 Bioanalyzer (Agilent Technologies B.V., The Netherlands). RNA was amplified using Illumina TotalPrep RNA Amplification Kit according to manufacturer’s instructions (ThermoFisher Scientific, Massachusetts, USA). A total of 750 ng cRNA was hybridized using the standard protocol from Illumina. Data was analyzed after background subtraction using GenomeStudio (V2010.1, Illumina), and processed with R2.10.1 lumi-package in R (30 (link)). Differentially expressed (DE) probes (adjusted p-value < 0.001) were identified when comparing to undifferentiated BMSCs using Iimma package from Bioconductor (31 (link)).