Microarray Analysis Using Amplified mRNA from Laser Capture Microdissection of Microscopic Hepatocellular Precancerous Lesions and Frozen Hepatocellular Carcinomas Reveals Unique and Consistent Gene Expression Profiles

Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
Toxicologic Pathology (Impact Factor: 2.14). 05/2003; 31(3):295-303. DOI: 10.1080/01926230390204333
Source: PubMed


The indirect labeling cDNA microarray technique was used to evaluate gene expression profiles of pure cell populations from frozen sections of carcinomas and adenomas harvested from precancerous hepatocellular lesions by using laser capture microdissection (LCM). The levels of differentially expressed genes were investigated using a cDNA microarray with 9,984 features with only 2 ug of two-round amplified aRNA, equivalent to 35 cells from LCM-adenomas and frozen samples of carcinomas from simian virus 40 (SV40) large T antigen transgenic rats. A total of 855 genes were identified as being 3-fold or more differentially expressed in carcinomas or adenomas as compared to normal tissue controls. Among these 855 genes, 71 genes were differentially expressed in both carcinomas and adenomas. Commonly up-regulated genes in both carcinoma and adenomas were 28 while 41 of the 71 genes were commonly down-regulated. Two genes, Igh1 (immunoglobulin heavy chain 1(Serum IgG2a), Image clone ID: 875880) and EST clone (AI893585, Image clone ID: 596604) were more than 7-fold up-regulated in carcinomas and 6-fold down-regulated in adenomas. In Cy5 and Cy3 reciprocal experiments for screening out false positive signals, the amplified carcinomas showed higher Pearson Correlation Coefficient values (-0.94 and -0.92) than the LCM-amplified adenoma samples (-0.79 and -0.84). LCM-amplified samples provided higher signal intensities over backgrounds and a greater average of Cy5:Cy3 ratios. Expression levels of mRNAs from selected genes, determined by using traditional dot blot analysis, revealed that 36 of 40 tested expression profiles were consistent with the microarray data. Thus, amplified aRNA harvested from homogeneous cell types using LCM can be applied to study gene expression profiles by use of microarray analysis.

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    • "This is most likely caused by the possible dilution of SV40-transformed cells with nontransformed cell types: LCM would ensure that RNA is only extracted from transformed enterocytes; villi fractionation would remove most of the muscle, mesenchyme, and crypt cells but still include goblet cells and enteroendocrine cells not expressing the SV40 T antigens; and SV40-transformed cells would be most dilute in whole intestine. LCM samples from liver were also shown to give more robust microarray ratios than samples obtained from dissected tissue (Yim et al., 2003). "
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