|
|
|
| Selection of Reliable Reference Genes Using Quantitative Real-time PCR in Caged Layers (Gallus gallus domesticus) During the Late Laying Period Under Different GluN Treatment |
| 2, 2, 2, 2, 2 |
|
|
|
|
Abstract The reference gene expression stability levels are the important factors to determine the reliability of quantitative Real-time polymerase chain reaction (qRT-PCR) results. This study was conducted to explore the selection of stable reference genes in different treatment of Glucosamine (GluN) to ensure the reliability and accuracy of gene expression analysis in caged layers (Gallus gallus domesticus) during the late laying period. A total of 500-day HY-Line layers were selected to the research objects which were randomly divided into four groups, the layers in control group were fed with the diet of the corn-soybean meal basal diet, while the others in experiment groups were fed the basal diet supplemented with 0.4%, 0.6%, 0.8% GluN, respectively. Expression of ten housekeeping genes, ribosomal protein S2 (RPS2), β-Actin, glyceraldehyde-3-phosphate dehydrogenase(GAPDH), hydroxymethyl biliary synthetase(HMBS), TATA-box binding protein(TBP), hypoxanthine phosphoribosyltransferase 1 (HPRT1), tubulin beta class (TUBB), succinate dehydrogenase complex flavoprotein subunit A (SDHA), ribosomal protein L4 (RPL4) and beta-2 microglobulin (B2M) were assessed by qRT-PCR in 8 tissues (heart, liver, lung, kidney, duodenum, pancreas, uterus and tibia), and 3 online reference genes stability assessment tools(geNorm, NormFinder, RefFinder) and the methods of Cycle threshold (Cq) and Delta cycle threshold (Delta CT) were used to the expression stability assessment of 10 reference gene by the qRT-PCR data. The results were shown as follows. The expression stability of same candidate reference gene varied with different treatment and tissues from the analysis of Cq mean value, which explained that the 10 reference genes had a certain expression difference in these 8 tissues. Under the different GluN treatment, the method of Delta CT found that the expression stability of reference genes were not the same, in general, the expression stability of TUBB、β-Actin and RPS2 were better than other genes. From the geNorm software, TBP/RPS2(1.08), RPS2/β-Actin(0.88), TBP/TUBB(1.16) and RPS2/HMBS(0.77) showed the stable expression under different GluN treatment, which showed that the expression stability of TBP, β-Actin and RPS2 were better. The NormFinder program software showed that RPS2 and HMBS were the stable genes. The refFinder online software showed that the RPS2 had most stable expression under 0.0% and 0.8% GluN group. From the comprehensive analysis of above results, RPS2 and TBP genes can apply to the quantitative expression analysis for the caged layers during the late laying period, while the expression stability of RPL4 gene was the worst, and it can not be applied to quantitative expression analysis. This experiment results also provide a theoretical basis for correcting the expression of target genes under the treatment of GluN.
|
|
Received: 12 August 2017
Published: 10 December 2017
|
|
|
|
|
|
|
