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

Capsule:
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.

Authors:
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

Abstract:

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 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.

Design:
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.

Setting:
Hospital.

Intervention (s):
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.

Results:
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.

Conclusion:
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.

  • Raul Gomez, PhD, IUIVI

    I firmly believe this is the kind of “preliminar” study anyone of us should perform when approaching the evaluation of gene regulation in a specific tissue. I wonder why we keep on “assuming” our HKG of choice is suitable for our studies instead of testing by ourselves or at least search the literature. Studies like this are of great importance to establish a wide data base which useful for all of us in order to select the best HKG for gene expression analysis with out need to test it by ourselves. Congrats again to the authors and all of those who perform similar studies like this, which help we all scientist to simplify the performing of our gene regulation studies

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