Variation in stability of housekeeping genes in healthy and adhesion related mesothelium
The most suitable housekeeping genes within healthy and adhesion-mesothelial tissue are ACTB, YWHAZ, and CYC1, as they are most stable in both tissue types.
Khaled Hassan Sadek, M.B.B.S., Felino Ramon Cagampang, Ph.D., Kimberley Davina Bruce, Ph.D., Nick Macklon, M.D., Ying Cheong, M.D.
Volume 98, Issue 4, Pages 1023-1027, October 2012
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 PCR (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 stability may be tissue specific and affected by certain pathologies.
Paired mesothelial (n=10) and adhesion tissue samples (n=10) were taken from during laparoscopic surgery. The stability of twelve candidate reference genes in the mesothelial tissues were evaluated; these includes ATP5b, SDHA, CYC1, 18S rRNA, RPL13A, ACTB, YWHAZ, TOP1, UBC, EIF4A2, GAPDH and B2M.
Assessment of HKG expression stability using geNorm algorithm software.
Main Outcome Measures:
Stability measure (M) generated by geometric averaging of multiple target genes and mean pair-wise variation of genes.
The most stable HKGs observed across both healthy and adhesion-related mesothlium were found to be ACTB, YWHAZ and CYC1. ACTB had a higher expression in healthy mesothelium compared with peritoneal adhesion tissue.
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 in inappropriate HKG selection.