Extraction and processing of high quality RNA from impalpable and macroscopically invisible prostate cancer for microarray gene expression analysis.
Molecular analyses of early-stage prostate cancers are necessary to assess their potential clinical significance based on established and/or novel biomarkers for tailored clinical management. A prerequisite for the application of RNA-based analyses of such, mostly macroscopically-undetectable, small prostate carcinomas is the recovery and preservation of sufficient RNA quantities and quality. Furthermore, in prostate cancer, heterogeneity is a common phenomenon that includes a juxtaposition of different tissue compositions and variable histological grades within the same tumor focus. To better understand the molecular mechanisms of prostate cancer, it is essential to correlate molecular data with a specific cell type. Here, we present a tissue collecting protocol which is aligned with the preoperative evaluation of tumor localization. In combination with the technique of laser microdissection and pressure catapulting, we are able to preserve RNA of high quality from homogeneous cell populations of macroscopically-undetectable small prostate carcinomas. To obtain the necessary RNA quantities for whole genome cDNA microarrays, the isolated total RNAs were amplified by T7-based RNA-polymerase in vitro transcription. The microarray analyses (Human Unigene Set RZPD3.1) resulted in 216 differentially expressed genes (191 down-regulated, 25 up-regulated). Among these were several known prostate cancer relevant genes, such as AMACR, TARP, LIM, GPR160 (all up-regulated), CAV1, NTN1, MT1X; CLU, TRIM29, SPARCL1 and HSPB8 (all down-regulated).
Schlomm
Thorsten
T
Department of Urology, University Clinic Hamburg-Eppendorf, D-20246 Hamburg, Germany. tschlomm@uke.uni-hamburg.de
Luebke
Andreas M
AM
Sültmann
Holger
H
Hellwinkel
Olaf J C
OJ
Sauer
Ulrich
U
Poustka
Annemarie
A
David
Kerstin A
KA
Chun
Felix K H
FK
Haese
Alexander
A
Graefen
Markus
M
Erbersdobler
Andreas
A
Huland
Hartwig
H
D015536:Down-Regulation / Q000235:genetics; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D008297:Male; D009367:Neoplasm Staging; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015854:Up-Regulation / Q000235:genetics
D012334:RNA, Neoplasm