How useful are housekeeping genes? Variable expression in melanoma metastases.
BACKGROUND
There is a certain difference in opinion regarding the optimal choice of housekeeping genes used as normalization factors in gene expression analysis. We have therefore examined the suitability of three housekeeping genes, hypoxanthine phosphoribosyl transferase, beta-glucuronidase and beta2-micro-globulin, for normalization of expression data from melanoma metastases.


METHODS
The expression of the three housekeeping genes was quantified by quantitative reverse transcription PCR in snap-frozen sections from 44 melanoma metastases, of which 19 were from patients treated with cisplatinum, dacarbazine and interferon-alpha2b.


RESULTS
The expression of each housekeeping gene varied considerably between the different metastases. Histopathological examination of the tissue sections revealed variation in the amount of tumor cells in the tissue, necrosis, varying degrees of lymphocyte infiltration, and lymph node remnants. Based on this examination, 16 biopsies were omitted from further analysis because they had cracked, contained empty or necrotic areas, or were dominated by lymph node tissue. Even in sections with more than 90% tumor cells, a wide variation in the expression of the three housekeeping genes was found. The amount of lymphatic infiltrate in the tumors can have an effect on the expression of housekeeping genes in the metastases, whereas treatment did not seem to influence the expression.


CONCLUSIONS
We conclude that the choice of housekeeping genes can have great impact on the normalization of specific genes in melanoma metastases. Furthermore, in the analysis of mRNA expression in tumor tissue, microscopic examination is of great importance to evaluate the integrity and cellular composition of the biopsy.
Kågedal
Bertil
B
Department of Clinical and Experimental Medicine, Division of Clinical Chemistry, Linköping University, Linköping, Sweden. bertil.kagedal@lio.se
Farnebäck
Malin
M
Håkansson
Annika
A
Gustafsson
Bertil
B
Håkansson
Leif
L
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D020043:Genes, Essential; D006801:Humans; D008297:Male; D008545:Melanoma / Q000235:genetics* / Q000473:pathology; D008875:Middle Aged; D009362:Neoplasm Metastasis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics
D012333:RNA, Messenger