Methodological approach to study the genomic profile of the human breast

Abstract

Abstract.One of the key end-points for understanding the molecular basis of the breast in its normal and cancer status is the quantitation of gene expression in specific cell populations. Microdissection techniques allow extraction of morphologically distinct cells for molecular analysis. The objective of this study was to determine the optimal RNA isolation and amplification to perform genomic expression analysis using the microarray technique from normal breast paraffin-embedded tissue samples using laser capture microdissection (LCM). We isolated epithelial and interlobular stroma cells from normal breast tissue and the total RNA was amplified using a PCR methodology developed by us, and in parallel the same starting material was used for amplification using the linear methodology. After two rounds of RNA amplification, we checked the quality of each amplified RNA and carried out the hybridization with cDNA glass-microarrays employing 15,000 genes for each replicate. In conclusion, we have successfully demonstrated that our PCR methodology is accurate and precise and give us a higher yield of amplified RNA from small number of cells obtained from LCM compared with the typical linear amplification methodology.

Full-term Pregnancy Induces a Specific Genomic Signature in the Human Breast

“…Cluster C represents differentiation-associated genes whose level of expression continuously and progressively increases with time of pregnancy, reaching their highest levels between 21 and 42 days postpartum, and cluster D comprises genes that are up-regulated around the 15th day of pregnancy and become progressively down-regulated from the end of pregnancy until the 42nd day postpartum (25). These observations confirm at genomic level our previous morphologic and physiologic findings indicating that temporal and sequential changes have to occur in the development of the mammary gland for accomplishing a protective degree of differentiation (11,12,(25)(26)(27)(28). The importance of identifying a specific signature by 42 days postpartum is highlighted by the observations that administration of the polycyclic hydrocarbon 7,12-dimethylbenz(a)anthracene to parous rats results in a markedly reduced tumorigenic response, supporting the concept that the differentiation induced by pregnancy shifts the susceptible ''intermediate cells '' that originate mammary cancer in the terminal end buds of the virginal gland (5, 10) to transformation-resistant cells (11,12).…”

“…Normal-appearing tissues were taken from areas at a distance z2 cm from any grossly identifiable lesion and immediately fixed in 70% ethanol for 8 h, followed by dehydration, paraffin embedding, sectioning, and staining for histologic analysis and laser capture microdissection following previously described procedures (28). Histopathologic diagnosis of tumor type was made by pathologists at each site (Table 1).…”

“…Mirror glass slides were used for robotically spotting 40,000 cDNAs representing 28,000 distinct human transcripts, 10,000 identified by expressed sequence tags, and 2,000 controls and blank spots. Probe construction using direct labeling with random hexamer primer and purification using the QIA-quick PCR purification kit (Qiagen) were done as previously described (28). After the last centrifugation at 13,000 rpm for 1 min, the concentration of the eluted material was determined, then partially dried in a vacuum centrifuge and resuspended in 15 AL of hybridization buffer containing 20Â SSC and 0.6 AL of 10% (w/v) SDS.…”

“…RNA isolation and amplification from laser capture microdissection samples were done as previously described (28). Microarrays were prepared by the Fox Chase Cancer Center National Cancer Institute -supported Microarray Facility.…”

“…Each gene was analyzed in triplicate, normalized against h-actin, and expressed in relation to a calibrator sample. Results were expressed as relative gene expression using the DC t method, as previously described (28).…”