TY - JOUR
T1 - Tissue-specific selection of reference genes is required for expression studies in the mouse model of maternal protein undernutrition
AU - Lucas, E.S.
AU - Watkins, A.J.
AU - Cox, A.L.
AU - Marfy-Smith, S.J.
AU - Smyth, N.
AU - Fleming, T.P.
PY - 2011/8
Y1 - 2011/8
N2 - Suboptimal maternal nutrition during gestation results in the establishment of long-term phenotypic changes and an increased disease risk in the offspring. To elucidate how such environmental sensitivity results in physiological outcomes, the molecular characterisation of these offspring has become the focus of many studies. However, the likely modification of key cellular processes such as metabolism in response to maternal undernutrition raises the question of whether the genes typically used as reference constants in gene expression studies are suitable controls. Using a mouse model of maternal protein undernutrition, we have investigated the stability of seven commonly used reference genes (18s, Hprt1, Pgk1, Ppib, Sdha, Tbp and Tuba1) in a variety of offspring tissues including liver, kidney, heart, retro-peritoneal and inter-scapular fat, extra-embryonic placenta and yolk sac, as well as in the preimplantation blastocyst and blastocyst-derived embryonic stem cells. We find that although the selected reference genes are all highly stable within this system, they show tissue, treatment and sex-specific variation. Furthermore, software-based selection approaches rank reference genes differently and do not always identify genes which differ between conditions. Therefore, we recommend that reference gene selection for gene expression studies should be thoroughly validated for each tissue of interest.
AB - Suboptimal maternal nutrition during gestation results in the establishment of long-term phenotypic changes and an increased disease risk in the offspring. To elucidate how such environmental sensitivity results in physiological outcomes, the molecular characterisation of these offspring has become the focus of many studies. However, the likely modification of key cellular processes such as metabolism in response to maternal undernutrition raises the question of whether the genes typically used as reference constants in gene expression studies are suitable controls. Using a mouse model of maternal protein undernutrition, we have investigated the stability of seven commonly used reference genes (18s, Hprt1, Pgk1, Ppib, Sdha, Tbp and Tuba1) in a variety of offspring tissues including liver, kidney, heart, retro-peritoneal and inter-scapular fat, extra-embryonic placenta and yolk sac, as well as in the preimplantation blastocyst and blastocyst-derived embryonic stem cells. We find that although the selected reference genes are all highly stable within this system, they show tissue, treatment and sex-specific variation. Furthermore, software-based selection approaches rank reference genes differently and do not always identify genes which differ between conditions. Therefore, we recommend that reference gene selection for gene expression studies should be thoroughly validated for each tissue of interest.
KW - reference genes
KW - maternal diet
KW - mouse embryonic stem cells
KW - mouse embryos
KW - real-time qPCR
KW - gene expression
UR - http://www.scopus.com/inward/record.url?scp=79959839456&partnerID=8YFLogxK
U2 - 10.1016/j.theriogenology.2011.03.008
DO - 10.1016/j.theriogenology.2011.03.008
M3 - Article
AN - SCOPUS:79959839456
SN - 0093-691X
VL - 76
SP - 558
EP - 569
JO - Theriogenology
JF - Theriogenology
IS - 3
ER -