In brief, 10 ng of total RNA isolated with the mirVana miRNA Isolation Kit was subjected to cDNA sysnthesis using microRNA specific stem-loop primer.
cRNA probes were synthesized and labeled using GeneChip Whole Transcript cDNA Synthesis and Amplification Kit and Terminal Labeling Kit (Affymetrix), and were subjected to hybridization with GeneChip Human Gene 1.0 ST Array (Affymetrix) that contains 28,869 well annotated genes.
After measuring protein concentration using the Bradford assay kit (Bio0Rad), lysates were subjected to ELISA.
Plasmids were purified using the mini prep kit (Qiagen) and were subjected to sequencing reactions using the ABI BigDye Terminator BDT 3.1 (Applied Biosystems, Foster City, CA, USA).
Recombinant plasmid DNAs were purified using the EndoFree Plasmid Maxi Kit (Qiagen) and were subjected to nucleotide sequencing to verify their authenticity.
The sequencing reactions (BigDye Terminator v. 3.1 Ready Reaction Cycle Sequencing Kit; Applied Biosystems, Foster City, CA) were subjected to purification using BigDye Xterminator (Applied Biosystems, Foster City, CA) for a complete removal of ddNTPs and underwent capillary electrophoresis on an Applied Biosystems 3130xl DNA Analyzer.
The amplified products were purified with Qiagen PCR Purification Kit (Qiagen) and were subjected to a second round of amplification with a 5′UTR forward primer with a NotI restriction site (5′-ataagaatgcggccgcGTC CGT CGC TGC TTC GGT G-3′) and a 3′UTR reverse primer with a BamHI restriction site (5′-atacgcggatccGCC TGT GAT GCG TGA TGA CG-3′).
The peptides were labeled with iTRAQ-114 (basal xenograft 1), iTRAQ-115 (basal xenograft 2), iTRAQ-116 (luminal xenograft 1), and iTRAQ-117 (luminal xenograft 2) reagents from a 4-plex iTRAQ reagent kit, and they were subjected to LC-MS/MS analysis.
50 μg protein, calculated using a BCA (Bicinchoninic Acid) Protein Assay Kit (Pierce, Rockford, IL), were subjected to SDS-PAGE.
DNA samples (500 ng) obtained from peripheral blood cells were bisulfite-converted using the EZ-96 DNA Methylation™ Kit (Zymo Research, Irvine, CA) and were subjected to methylation profiling using the Illumina Infinium HumanMethylation450 BeadChip assay (Illumina, San Diego, CA).
