Results were determined by quantitative RT-PCR and normalized to ywhaz
(housekeeping gene) and were performed by the [DELTA][DELTA]CT method.
Other primers used in this study were as follows: YWHAZ
(forward): 5'- TGTTGTAGGAGCCCGTAG-3' and YWHAZ
(reverse): 5'- GACCCAGTCTGATAGGATGT-3', GADPH (forward) 5'- GAAGGTGAAGGTCGGAGT-3' and GADPH (reverse): 5'-GAAGATGGTGATGGGATTTC-3'.
The expression data for each gene was divided by that for the YWHAZ
Ding et al., "MicroRNA-451 regulates p38 MAPK signaling by targeting of Ywhaz
and suppresses the mesangial hypertrophy in early diabetic nephropathy," The FEBS Letters, vol.
The following genes were selected for validation from a set of novel highly significant upregulated genes from our expression dataset (Table 3): DFFA, RIOK3, E2F5, EIF3H, YWHAZ
, QSER1, RPS6KA3, PRPF38A, MCM7, and C20ORF27.
miR-451 negatively regulated the expression of its target gene Ywhaz
, which was required for the activation of p38 MAPK signaling, and inhibited glomerular mesangial cell proliferation in early DN, thus preventing mesangial hypertrophy.[sup]
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ
), hypoxanthine phos-phoribosyltransferase 1 (HPRT1), and RPLP0 were selected via geNorm and Normfinder as reference genes to normalize the data.
UBC and YWHAZ
as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116and HepG2cell lines.
For instance, RPL4 and TBP ranked as the two most stably expressed genes in PBMC of pigs at day 1, day 7, day 14 after heat stress, while B2M was the most stable at day 21 after heat stress (Xiang-Hong et al., 2011); PPIA, B2M, and RPL4 were the most stable reference genes in Lipopolysaccharide (LPS) stimulated PBMC, while Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ
), RPL4 and PPIA were the most stably expressed reference genes in the case of lipoteichoicacid (LTA) stimulated PBMC (Cinar et al., 2012; Cinar et al., 2013).
The ectopic expression of miR-375 reduced cell viability and induced apoptosis by targeting PDK1 and YWHAZ
Gene expression was normalized to the geometric mean of three housekeeping genes (HPRT, TBP, and YWHAZ
for BeWo cells; HPRT, GAPDH, and YWHAZ
for primary CT cells).
In Table 2, a highly conserved monooxygenase-associated protein YWHAZ
was identified as a functional protein in intestinal bowel disease.