Gene expression profile of bone marrow from MMTV-NeuT mouse model of spontaneous mammary carcinogenesis.

We investigated, in a spontaneous mouse model of breast carcinogenesis (MMTV/NeuT), the modifications in the hematopoietic and stromal bone marrow (BM) compartments and their correlation with the pathological stage of the primary mammary lesions, following the idea that signs of the cross-talk between the BM and elements located in the tumor microenvironment, either neoplastic or stroma/immune cells, can be identified in the very early phases of cancer development, and lead to the instruction of a tumor-promoting hematopoiesis. Total RNA was extracted from BM cells of MMTV-NeuT transgenic females, and age-matched non transgenic siblings, at 6, 12 and 24 weeks of age, using Trizol reagent (Life Technologies) following the manufacturer’s instruction. RNA integrity was verified using the RNA 6000 Nano Kit (Agilent Technologies, Palo Alto, USA). All samples had an RNA integrity number (RIN) > 7, indicating good quality. For gene expression profile (GEP), RNA was reverse transcribed, labeled with biotin and amplified overnight using the Illumina TotalPrep RNA Amplification Kit (Life Technologies). Biotinylated complementary RNA was hybridized to mouseWG-6 v2.0 Expression BeadChips (Illumina, San Diego, USA). Illumina mouseWG-6 v2.0 Expression BeadChips microarrays were scanned with Illumina BeadArray Reader, and raw data obtained using Illumina BeadStudio v3.3.8 were processed using the lumi package from R/Bioconductor. Raw data were log2-transformed, normalized using robust spline normalization and filtered; only the probes with a detection p-value <0.01 in at least one sample were further analysed.