Endogenous control genes in prostate cells: evaluation of gene expression using 'real-time' quantitative polymerase chain reaction.
OBJECTIVE
Our aims were to measure the level of expression of Abelson (ABL1), β-glucuronidase (GUS) and glucose-6-phosphate dehydrogenase (G6PD) genes in exfoliated urine cells from healthy and transrectal ultrasound biopsy patients with elevated prostate-specific antigen levels and/or abnormal digital rectal examinations or urinary symptoms indicative of prostate problems, as well as in archived formalin-fixed paraffin-embedded prostate materials.


METHODS
Real-time quantitative polymerase chain reaction (RQ-PCR) was used to evaluate the suitability of the 3 control genes, i.e. ABL1, GUS and G6PD, as control genes for prostate cancer cells. Exfoliated urine cells from 30 healthy males, 53 male patients, 138 cases of archived paraffin-embedded prostate tissues and 3 prostate cell lines were sampled. All cells were lysed in guanidine isothiocyanate buffer from which RNA was extracted and converted to cDNA by random hexamer priming. RQ-PCR was performed using TaqMan chemistries.


RESULTS
There was no significant difference in the level of expression for each of the 3 control genes in the cell lines. There was a significant difference in GUS transcript level between patients and healthy controls in both urine and prostate tissue sections (p < 0.05). G6PD transcript numbers also differed significantly from those of GUS in the prostate cell lines and tissue sections (p < 0.05). The transcript numbers of all the control genes were significantly reduced in aged samples (p < 0.001).


CONCLUSIONS
The ABL1 gene was the most stable control gene in both clinical specimens and cell lines. Therefore, we recommend its use to enable standardization and interlaboratory comparisons for the RQ-PCR of prostatic tumour markers.
Nna
Emmanuel
E
Cranfield Health, Cranfield University, Cranfield, UK. e.nna@safetybiomedical.org
Tothill
Ibtisam E
IE
Ludeman
Linmarie
L
Bailey
Tracey
T
D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis* / Q000235:genetics; D033942:Adaptor Proteins, Vesicular Transport; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D005954:Glucosephosphate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D005966:Glucuronidase / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D011467:Prostate / Q000166:cytology / Q000378:metabolism*; D017430:Prostate-Specific Antigen / Q000032:analysis; D011470:Prostatic Hyperplasia / Q000175:diagnosis / Q000473:pathology; D011471:Prostatic Neoplasms / Q000175:diagnosis / Q000473:pathology; D011544:Pseudogenes; D013048:Specimen Handling
C487178:AHI1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D014408:Biomarkers, Tumor; D005954:Glucosephosphate Dehydrogenase; D005966:Glucuronidase; D017430:Prostate-Specific Antigen