Variation in stability of housekeeping genes in healthy and adhesion-related mesothelium

Objective: To investigate the stability of various housekeeping genes (HKG) within healthy versus scarred peritoneal mesothelium. The use of HKG as internal controls for quantitative real-time polymerase chain reaction (qRT-PCR) studies is based on the assumption of their inherent stability. However, recent evidence suggests that this is not true for all HKG and that stability may be tissue specific and affected by certain pathologies. Design: Paired mesothelial (n = 10) and adhesion tissue samples (n = 10) were taken during laparoscopic surgery. The stability of 12 candidate reference genes in the mesothelial tissues were evaluated; these include ATP5b, SDHA, CYC1, 18S rRNA, RPL13A, ACTB, YWHAZ, TOP1, UBC, EIF4A2, GAPDH, and B2M. Setting: Hospital. Patient(s): Female patients undergoing laparoscopic gynecological surgery were recruited from the Princess Anne Hospital, United Kingdom. Intervention(s): Assessment of HKG expression stability using geNorm algorithm software. Main Outcome Measure(s): Stability measure (M) generated by geometric averaging of multiple target genes and mean pairwise variation of genes. Result(s): The most stable HKGs observed across both healthy and adhesion-related mesothelium were found to be ACTB, YWHAZ, and CYC1. ACTB had a higher expression in healthy mesothelium compared with in peritoneal adhesion tissue. Conclusion(s): This study indicates that ACTB, YWHAZ, and CYC1 are the appropriate internal controls for qRT-PCR analysis in mesothelial gene expression studies. Published discrepancies in gene expression studies using the mesothelium may therefore be due in part to inappropriate HKG selection.