GAPDH, 18S rRNA and YWHAZ are suitable endogenous reference genes for relative gene expression studies in placental tissues from human idiopathic fetal growth restriction.
Comparative gene expression studies in the placenta may provide insights into molecular mechanisms of important genomic alterations in pregnancy disorders. Endogenous reference genes often referred to as housekeeping genes, are routinely used to normalise gene expression levels. For this reason, it is important that these genes be empirically evaluated for stability between placental samples including samples from complicated pregnancies. To address this issue, six candidate housekeeping genes including several commonly used ones (ACTB, GAPDH, 18S rRNA, TBP, SDHA and YWHAZ) were investigated for their expression stability in placentae obtained from pregnancies complicated by idiopathic FGR (n=25) and gestation-matched control pregnancies (n=25). Real-time PCR was performed using pre-validated gene expression assay kits. The geNorm program was used for gene stability measure (M) for the entire housekeeping genes in all control and FGR-affected placental samples. Results showed that GAPDH and 18S rRNA were most stable, with an average expression stability of M=0.441 and 0.443, respectively, followed by YWHAZ (M=0.472). SDHA, ACTB and TBP were the least stable housekeeping genes (M=0.495, 0.548 and 1.737, respectively). We recommend geometric averaging of two or more housekeeping genes to determine relative gene expression levels between FGR-affected and control placentae.
Murthi
P
P
Pregnancy Research Centre, Department of Perinatal Medicine, The Royal Women's Hospital, University of Melbourne, Carlton, Victoria 3053, Australia. padma@unimelb.edu.au
Fitzpatrick
E
E
Borg
A J
AJ
Donath
S
S
Brennecke
S P
SP
Kalionis
B
B
D000328:Adult; D005260:Female; D005317:Fetal Growth Retardation / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000096:biosynthesis*; D006801:Humans; D010920:Placenta / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D012337:RNA, Ribosomal, 18S / Q000096:biosynthesis*
D012337:RNA, Ribosomal, 18S; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)