cDNA microarray analysis with amplified RNA after isolation of intact cellular RNA from neoplastic and non-neoplastic prostate tissue separated by laser microdissections.
Laser microdissection is a valuable tool to prepare pure cell populations from complex tissues for further analyses. Gene expression studies by real-time RT-PCR and cDNA arrays of microdissected tissues are becoming widely used methods. The integrity and quantity of prepared RNA must be proven to ensure reliable results in subsequent applications such as quantitative RT-PCR and cDNA-arrays. In the present study we used RNAlater trade mark protected prostate tissue for laser microdissection of tumor and tumor-free tissues. RNA quality and quantity was assessed using automated capillary gel electrophoresis. By using quantitative real time-RT-PCR before and after mRNA amplification the insulin-like growth factor binding protein-3 (IGFBP-3) gene expression was shown to be down-regulated in three out of five cases and DD3 was up-regulated in cancer tissues in all cases. The up-regulation of DD3 and the down-regulation of IGFBP-3 gene expression in cancer tissue were conserved after RNA amplification. A cDNA microarray also revealed an IGFBP-3 down-regulation in cancer tissue as well as several genes known to be differerentially expressed in prostate cancer. Taken together, we present a novel method for the isolation of intact RNA from laser microdissection-derived prostate cancer tissue useful for downstream applications of real-time RT-PCR and cDNA microarrays.
Thelen
Paul
P
Department of Urology, Georg-August-Universität, D-37075 Göttingen, Germany. pthelen@gwdg.de
Burfeind
Peter
P
Grzmil
Michal
M
Voigt
Silke
S
Ringert
Rolf-Hermann
RH
Hemmerlein
Bernhard
B
D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics* / Q000378:metabolism; D008297:Male; D020411:Oligonucleotide Array Sequence Analysis; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000951:Antigens, Neoplasm; D018972:Insulin-Like Growth Factor Binding Protein 3; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C479341:prostate cancer antigen 3, human