Balogh Gabriela A, Heulings Rebecca, Mailo Daniel, Wang Richard, Li Yue-Sheng, Hardy Randy, Russo Jose
Breast Cancer Research laboratory, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Int J Oncol. 2007 Aug;31(2):253-60.
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.
了解乳腺正常和癌症状态分子基础的关键终点之一是特定细胞群体中基因表达的定量。显微切割技术可提取形态学上不同的细胞用于分子分析。本研究的目的是确定使用激光捕获显微切割(LCM)从正常乳腺石蜡包埋组织样本中,采用微阵列技术进行基因组表达分析的最佳RNA分离和扩增方法。我们从正常乳腺组织中分离出上皮细胞和小叶间基质细胞,使用我们开发的PCR方法扩增总RNA,同时使用线性方法对相同的起始材料进行扩增。经过两轮RNA扩增后,我们检查了每个扩增RNA的质量,并使用包含15,000个基因的cDNA玻璃微阵列进行杂交,每个重复样本都进行了检测。总之,我们成功证明了我们的PCR方法准确且精确,与典型的线性扩增方法相比,从LCM获得的少量细胞中能产生更高产量的扩增RNA。