Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer.
BACKGROUND
To discover prostate cancer biomarkers, we profiled gene expression in benign and malignant cells laser capture microdissected (LCM) from prostate tissues and metastatic prostatic adenocarcinomas. Here we present methods developed, optimized, and validated to obtain high quality gene expression data.


RESULTS
RNase inhibitor was included in solutions used to stain frozen tissue sections for LCM, which improved RNA quality significantly. Quantitative PCR assays, requiring minimal amounts of LCM RNA, were developed to determine RNA quality and concentration. SuperScript II reverse transcriptase was replaced with SuperScript III, and SpeedVac concentration was eliminated to optimize linear amplification. The GeneChip(R) IVT labeling kit was used rather than the Enzo BioArray HighYield RNA transcript labeling kit since side-by-side comparisons indicated high-end signal saturation with the latter. We obtained 72 mug of labeled complementary RNA on average after linear amplification of about 2 ng of total RNA.


CONCLUSIONS
Unsupervised clustering placed 5/5 normal and 2/2 benign prostatic hyperplasia cases in one group, 5/7 Gleason pattern 3 cases in another group, and the remaining 2/7 pattern 3 cases in a third group with 8/8 Gleason pattern 5 cases and 3/3 metastatic prostatic adenocarcinomas. Differential expression of alpha-methylacyl coenzyme A racemase (AMACR) and hepsin was confirmed using quantitative PCR.
Kube
Dagmar M
DM
Department of Laboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, USA. Kube@fr.com <Kube@fr.com>
Savci-Heijink
Cemile D
CD
Lamblin
Anne-Françoise
AF
Kosari
Farhad
F
Vasmatzis
George
G
Cheville
John C
JC
Connelly
Donald P
DP
Klee
George G
GG
D005784:Gene Amplification; D020869:Gene Expression Profiling; D005819:Genetic Markers; D006801:Humans; D007834:Lasers; D008297:Male; D042282:Microdissection; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D012334:RNA, Neoplasm / Q000235:genetics*; D014158:Transcription, Genetic
D005819:Genetic Markers; D012334:RNA, Neoplasm