The Analysis Of Gene Expression Profile In Placentaomes From Transgenic SCNT Bovine And QPCR Validation

SCNT is often used to produce transgenic animals. Although cattles have the bestsuccess rate of cloning pregnancy, the overall success rante is only10%of naturalreproduction or artificial fertilization. Studies have improved that placental abnormaldevelopment is a key reason of the low success rate of cloning pregnancy. Therefore,Somatic cell nuclear transfer technology is often used as a platform to produce transgenictransgenic animals. In all cloned species, the cattle has the highest pregnancy success rates, but theoverall success rante do not exceed10%success rate of the natural or artificial inseminationpregnancy. Studies have confirmed that placental abnormalities are a major cause of low pregnancysuccess rate in cloned animals. Therefore, it is particularly important to focus on the molecularmechanism of cloned placental abnormalities. A comprehensive analysis of the gene expressionprofiles may offer theoretical molecular basis to solve the abnormal placental development of thecloned cattle.In this study, two experimental groups were placentomes of two transgenic SCNT, and acontrol group was placentomes from a natural reproduced cattl. Transcriptome sequencing (RNA-Seq) for the three samples was performed by the second-generation sequencing technology. Theresults were normalized by the RPKM treatment and then were clustered by functional analysis(GO and KEGG Pathway). It showed that up-regulated genes were enriched in cell adhesion,extracellular matrix function and down-regulatedgenes were mainly enriched in immune relatedactivities. This may indicate that the transgenic cloned cattle used in this study were abnormal.Limited by reducing of cell migration, the crosstalk between fetus and matrix was negativeaffected. In addition, the down-regulated genes were associated with immune activities in thetransgenic cloned bovine placentas, which increased the risk of infection.RNA-Seq analysis of differentially expressed genes has no internal reference, so it is likelyoccurring false positive errors. Therefore, this study validated the differentially expressed genes byquantitative PCR, and based on sequencing results and quantitative results we can determine theconsistency degree of the two results. But validating all the differentially expressed genes by quantitative PCR assay is unrealistic. So the validating arrangement should be narrow down, andthe method must be scientific and responsible. In this study, the results mainly refered the result ofsignaling pathways, and partly refered the results of gene act network, GO analysis, special genes.After that, there were30genes were selected for qPCR validation. Based the validation results,more than eighty percent of key genes were consistent quantitative results with sequencing results,which suggested the sequencing result was very good. Meanwhile, the primers in this study couldincreased the bovine primer data.Particularly, the GAPDH gene is often used as a reference gene for qPCR because its stableexpressesion the volume is very, for relative expression of other genes. However, both sequencingand quantitative validation improved that GAPDH expression levels in the experimental group hassignificantly changed in this study, which suggests GAPDH may not suitable as a reference genefor qPCR in term of cloned bovine study..