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

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.

Keywords

cDNA microarray indirect labeling amplified mRNA laser capture microdissection.

References

Bonner RF , Emmert-Buck M. , Cole K. , Pohida T. , Chuaqui R. , Goldstein S. , Liotta LA (1997). Laser capture microdissection: Molecular analysis of tissue. Science 278: 1481—1483.

Daigo Y. , Chin SF , Gorringe KL , Bobrow LG , Ponder BAJ , Pharoah PDP , Caldas C. (2001). Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers: A new approach for the molecular analysis of paraffin-embedded cancer tissue. Am J Pathol 158: 1623—1631.

Dragan Y. , Valdes R. , Gomez-Angelats M. , Felipe A. , Javier CF , Pitot H. , Pastor-Anglada M. (2000). Selective loss of nucleoside carrier expression in rat hepatocarcinomas. Hepatology 32: 239—246.

Eberwine J. , Yeh H. , Miyashiro K. , Cao Y. , Nair S. , Finnell R. , Zettel M. , Coleman P. (1992). Analysis of gene expression in single live neurons . Proc Natl Acad Sci USA 89: 3010—3014.

Emmert-Buck MR , Bonner RF , Smith PD , Chuaqui RF , Zhuang Z. , Goldstein SR , Weiss RA , Liotta LA (1996). Laser capture microdissection . Science 274: 998—1001.

Gannot G. , Gannot I. , Vered H. , Buchner A. , Keisari Y. (2002). Increase in immune cell infiltration with progression of oral epithelium from hyperkeratosis to dysplasia and carcinoma. Br J Cancer 86: 1444—1448.

Haines DC , Chattopadhyay S. , Ward JM ( 2001). Pathology of aging B6;129 mice. Toxicol Pathol 29: 653—661.

Hully JR , Su Y. , Lohse JK , Griep AE , Sattler CA , Haas MJ , Dragan Y. , Peterson J. , Neveu M. , Pitot HC (1994). Transgenic hepatocarcinogenesis in the rat. Am J Pathol 145: 386—397.

Mills JC , Roth KA , Cagan RL , Gordon JI (2001). DNA microarrays and beyond: Completing the journey from tissue to cell. Nat Cell Biol 3: E175—E178.

10.

Perou CM , Sorlie T. , Eisen MB , van de RM , Jeffrey SS , Rees CA , Pollack JR , Ross DT , Johnsen H. , Akslen LA , Fluge O. , Pergamenschikov A. , Williams C. , Zhu SX , Lonning PE , Borresen-Dale AL , Brown PO , Botstein D. (2000). Molecular portraits of human breast tumours. Nature 406: 747—752.

11.

Sargent LM , Dragan YP , Sattler G. , Xu YH , Wiley J. , Pitot HC (1997). Specific chromosomal changes in albumin simian virus 40 T antigen transgenic rat liver neoplasms. Cancer Res 57: 3451—3456.

12.

Scheidl SJ , Nilsson S. , Kalen M. , Hellstrom M. , Takemoto M. , Hakansson J. , Lindahl P. (2002). mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures. Am J Pathol 160: 801—813.

13.

Sorlie T. , Perou CM , Tibshirani R. , Aas T. , Geisler S. , Johnsen H. , Hastie T. , Eisen MB , van de RM , Jeffrey SS , Thorsen T. , Quist H. , Matese JC , Brown PO , Botstein D. , Eystein LP , Borresen-Dale AL (2001). Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98: 10869—10874.

14.

Verma M. , Wright GL Jr, Hanash SM , Gopal-Srivastava R. , Srivastava S. (2001). Proteomic approaches within the NCI early detection research network for the discovery and identification of cancer biomarkers . Ann NY Acad Sci 945: 103—115.

15.

Wang E. , Miller LD , Ohnmacht GA , Liu ET , Marincola FM (2000). Highfidelity mRNA amplification for gene profiling . Nat Biotechnol 18: 457— 459.

16.

Xu XR , Huang J. , Xu ZG , Qian BZ , Zhu ZD , Yan Q. , Cai T. , Zhang X. , Xiao HS , Qu J. , Liu F. , Huang QH , Cheng ZH , Li NG , Du JJ , Hu W. , Shen KT , Lu G. , Fu G. , Zhong M. , Xu SH , Gu WY , Huang W. , Zhao XT , Hu GX , Gu JR , Chen Z. , Han ZG (2001). Insight into hepatocellular carcinogenesis at transcriptome level by comparing gene expression profiles of hepatocellular carcinoma with those of corresponding noncancerous liver. Proc Natl Acad Sci USA 98: 15089—15094.