Article

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: 1.92). 05/2003; 31(3):295-303. DOI: 10.1080/01926230390204333
Source: PubMed

ABSTRACT 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.

0 Bookmarks
 · 
159 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: To understand human pathology at the system level, one must examine the complex milieu of molecular interactions between various cells within human body, rather than the characteristics of isolated cell or cultured cell. However, all over the human body an understanding of the pathophysiology of particular tissues has lagged behind the insights in general biological processes. A “high-throughput revolution” unfolding in model clinical science leads to significant increase of knowledge describing transcriptome and proteome states in complex human diseases, including pathologies of the liver. Despite seeming accessibility of the profiling techniques, cautionary approach is necessary in the interpretation of the data. There are universal caveats that preclude meta-analysis of the data collected. Major biological obstacles are heterogeneity of the samples profiled, imperfect correlation between mRNA and protein levels, and normal variation in the levels of mRNAs and corresponding proteins in healthy individuals. Nevertheless, a significant gain in the understanding of NAFLD spectrum diseases reached recently is inseparable from the data accumulated in high-throughput projects.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The aim of this study was to identify genomic aberrations in hepatocellular carcinoma (HCC) by using laser capture microdissection (LCM) combined with microarray analysis. Samples were procured by LCM from HCC and patient-matched normal liver tissue surgically resected from 4 patients. RNA was isolated from the samples and reverse transcribed into cDNA. After 2-cycle linear amplification and 2-color fluorescent labeling, the cRNA was hybridized onto a whole genome microarray. All genes expressed in the normal and HCC samples were counted and analyzed. Differentially expressed genes were identified and the top 10 up and downregulated genes (totally 20 genes) were further evaluated. LCM was able to accurately capture 50-200 cells from HCC and control tissues. The microarray spectrum showed satisfactory detection of HCC-enriched genes. A total of 1361 differentially expressed genes were identified, among which, 607 were upregulated and 754 were downregulated. Among the top 20 up and downregulated genes, 4 genes had not been documented in the literature as being differentially expressed in any tumors. Thus, LCM is an effective approach for identifying aberrantly expressed genes in HCC, and may lead to the discovery of biomarkers for diagnostic and therapeutic applications.
    Genetics and molecular research: GMR 01/2013; 12(4):5527-36. DOI:10.4238/2013.November.18.3 · 0.85 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Microarrays have long been promised to be a tool that might one day revolutionize oncology research and drug development. Despite the tremendous potential, however, there have been few breakthroughs that can be directly attributed to microarray-based profiling. While many researchers now say that microarrays have been over-hyped, it is more likely that early experiments were simply conducted in a naïve manner. Many believe that as technology and our understanding of experimental design improves, so too will the end results. We believe that with new approaches, particularly the use of pure cell populations potentially coupled with new and improved RNA amplification methodologies, the promise of microarrays for oncology finally will be realized. Key WordsMicroarrays–gene expression–RNA amplification–laser capture microdissection–cancer stem cell theory–pure cell populations–diagnostics–prognostics
    12/2008: pages 3-18;

Full-text (2 Sources)

Download
101 Downloads
Available from
Jun 4, 2014