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.
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.
Female patients undergoing laparoscopic gynecological surgery were recruited from the Princess Anne Hospital, United Kingdom.
Assessment of HKG expression stability using geNorm algorithm software.
Stability measure (M) generated by geometric averaging of multiple target genes and mean pairwise variation of genes.
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.
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.
"This controls for variables such as the amount or quality of starting material, enzymatic efficiencies, and differences between tissues or cells in overall transcriptional activity (Vandesompele et al., 2002). We and others have previously shown that the expression level of these endogenous HKGs varies according to tissue type (Bruce et al., 2012; Hsiao et al., 2001; Sadek et al., 2012a, 2012b) and developmental stage (Sellayah et al., 2008; Warrington et al., 2000). They are constitutively expressed in the tissue and as they mediate basic cellular function were thought unlikely to vary due to the experimental conditions being investigated. "
[Show abstract][Hide abstract] ABSTRACT: The endogenous timing system within the suprachiasmatic nuclei (SCN) of the hypothalamus drives the cyclic expression of the clock molecules across the 24h day-night cycle controlling downstream molecular pathways and physiological processes. The developing fetal clock system is sensitive to the environment and physiology of the pregnant mother and as such disruption of this system could lead to altered physiology in the offspring. Characterizing the gene profiles of the endogenous molecular clock system by quantitative reverse transcription polymerase chain reaction is dependent on normalization by appropriate housekeeping genes (HKGs). However, many HKGs commonly used as internal controls, although stably expressed under control conditions, can vary significantly in their expression under certain experimental conditions. Here we analysed the expression of ten classic HKG across the 24h light-dark cycle in the SCN of mouse offspring exposed to normal chow or a high fat diet during early development and in postnatal life. We found that the HKGs glyceraldehyde-3-phosphate dehydrogenase, beta actin and adenosine triphosphate synthase subunit to be the most stably expressed genes in the SCN regardless of diet or time within the 24h light-dark cycle, and are therefore suitable to be used as internal controls. However SCN samples collected during the light and dark periods did show differences in expression and as such the timing of collection should be considered when carrying out gene expression studies.
Brain Research 05/2014; 1575(1). DOI:10.1016/j.brainres.2014.05.031 · 2.84 Impact Factor
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