Document Detail

Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?
MedLine Citation:
PMID:  16211407     Owner:  NLM     Status:  MEDLINE    
Using quantitative reverse transcription-polymerase chain reaction (RT-PCR), reference genes are utilized as endogenous controls for relative quantification of target genes in gene profiling studies. The suitability of housekeeping genes for that purpose in prostate cancer tissue has not been sufficiently investigated so far. The objective of this study was to select from a panel of 16 potential candidate reference genes the most stable genes for gene normalization. Expression of mRNA encoding ACTB, ALAS1, ALB, B2M, G6PD, GAPD, HMBS, HPRT1, K-ALPHA-1, POLR2A, PPIA, RPL13A, SDHA, TBP, UBC, and YWHAZ was examined in matched, microdissected malignant and nonmalignant tissue specimens obtained from 17 nontreated prostate carcinomas after radical prostatectomy by real-time RT-PCR. The genes studied displayed a wide expression range with cycle threshold values between 16 and 37. The expression was not different between samples from pT2 and pT3 tumors or between samples with Gleason scores <7 and >or=7 (P>0.05). ACTB, RPL13A, and HMBS showed significant differences (P<0.02 at least) in expressions between malignant and nonmalignant pairs. All other genes did not differ between the matched pairs, and the software programs geNorm and NormFinder were used to ascertain the most suitable reference genes from these candidates. HPRT1, ALAS1, and K-ALPHA-1 were calculated by both programs to be the most stable genes covering a broad range of expression. The expression of the target gene RECK normalized with HRPT1 alone and with the normalization factors generated by the combination of these three reference genes as well as with the unstable genes ACTB or RPL13A is given. That example shows the significance of using suitable reference genes to avoid erroneous normalizations in gene profiling studies for prostate cancer. The use of HPRT1 alone as a reference gene shown in our study was sufficient, but the normalization factors generated from two (HRPT1, ALAS1) or all three genes (HRPT1, ALAS1, K-ALPHA-1) should be considered for an improved reliability of normalization in gene profiling studies of prostate cancer.
Falk Ohl; Monika Jung; Chuanliang Xu; Carsten Stephan; Anja Rabien; Mick Burkhardt; Andreas Nitsche; Glen Kristiansen; Stefan A Loening; Aleksandar Radonić; Klaus Jung
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2005-10-07
Journal Detail:
Title:  Journal of molecular medicine (Berlin, Germany)     Volume:  83     ISSN:  0946-2716     ISO Abbreviation:  J. Mol. Med.     Publication Date:  2005 Dec 
Date Detail:
Created Date:  2005-12-02     Completed Date:  2006-05-03     Revised Date:  2011-07-08    
Medline Journal Info:
Nlm Unique ID:  9504370     Medline TA:  J Mol Med (Berl)     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  1014-24     Citation Subset:  IM    
Department of Urology, Charité-Universitätsmedizin Berlin, Campus Mitte, Schumannstrasse 20/21, 10098 Berlin, Germany.
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MeSH Terms
Carcinoma / genetics,  metabolism,  pathology
Gene Expression Profiling / methods,  standards*
Gene Expression Regulation, Neoplastic*
Middle Aged
Prostatic Neoplasms / genetics*,  metabolism,  pathology
RNA, Messenger / genetics,  metabolism*
Reference Standards
Reverse Transcriptase Polymerase Chain Reaction
Reg. No./Substance:
0/RNA, Messenger

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