Methodological approach to study the genomic profile of the human breast.
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.
Balogh
Gabriela A
GA
Breast Cancer Research laboratory, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Heulings
Rebecca
R
Mailo
Daniel
D
Wang
Richard
R
Li
Yue-Sheng
YS
Hardy
Randy
R
Russo
Jose
J
D001940:Breast / Q000473:pathology*; D001943:Breast Neoplasms / Q000235:genetics*; D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D015894:Genome, Human; D023281:Genomics / Q000379:methods*; D006801:Humans; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D017698:Postmenopause; D012334:RNA, Neoplasm
D017931:DNA Primers; D018076:DNA, Complementary; D012334:RNA, Neoplasm