Characterization of a method for profiling gene expression in cells recovered from intact human prostate tissue using RNA linear amplification.
Coupling array technology to laser capture microdissection (LCM) has the potential to yield gene expression profiles of specific cell populations within tissue. However, remaining problems with linear amplification preclude accurate expression profiling when using the low nanogram amounts of RNA recovered after LCM of human tissue. We describe a novel robust method to reliably amplify RNA after LCM, allowing direct probing of 12K gene arrays. The fidelity of amplification was demonstrated by comparing the ability of amplified RNA (aRNA) versus that of native RNA to identify differentially expressed genes between two different cell lines, demonstrating a 99.3% concordance between observations. Array findings were validated by quantitative polymerase chain reaction analysis of a randomly selected subset of 32 genes. Using LCM to recover normal (N=5 subjects) or cancer (N=3) cell populations from intact human prostate tissue, three differentially expressed genes were identified. Independent investigators have previously identified differential expression of two of these three genes, hepsin and beta-microseminoprotein, in prostate cancer. Taken together, the current study demonstrates that accurate gene expression profiling can readily be performed on specific cell populations present within complex tissue. It also demonstrates that this approach efficiently identifies biologically relevant genes.
Ding
Y
Y
Department of Medicine, Division of Hematology/Oncology, Northwestern University Medical School and the Robert H Lurie Cancer Center of Northwestern University, Chicago, IL, USA.
Xu
L
L
Chen
S
S
Jovanovic
B D
BD
Helenowski
I B
IB
Kelly
D L
DL
Catalona
W J
WJ
Yang
X J
XJ
Pins
M
M
Ananthanarayanan
V
V
Bergan
R C
RC
D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000473:pathology; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D042282:Microdissection / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011467:Prostate / Q000166:cytology; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D014158:Transcription, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
D012334:RNA, Neoplasm