Screening of siRNA for PTEN gene silencing in mouse primary hepatocytes
ZHOU Long1,2 WANG Li-lin3 HU Xu-huai1
1.Shenzhen Health Development Research Center,Guangdong Province,Shenzhen 518000,China；
2.Department of Anesthesiology,Shenzhen Maternity and Child Healthcare Hospital,Southern Medical University,Guangdong Province,Shenzhen 518000,China；
3.Shenzhen Blood Center,Guangdong Province,Shenzhen 518000,China
Abstract：Objective To screen siRNA for PTEN gene silencing in mouse primary hepatocytes.Methods Primary mouse hepatocytes were isolated by two-step collagenase perfusion,the hepatocytes were plated in collagen-coated 25 cm2 flasks at a density of 1×106cells/flask.The hepatocytes were divided into five groups on average：control group(C group),negative control group(NC group),siR-996 group,siR-1381 group,siR-1519 group.The siRNA of each group were transferred into mouse primary hepatocytes by HiPerFect transfection reagent,extracted RNA and protein after 24 h.The expression of PTEN mRNA was detected by Real-time PCR and the content of PTEN protein was detected by Western blot.Results The PTEN protein content of the siR-996 group,siR-1381 group and siR-1519 group were lower than that of the NC group,the differences were statistically significant(P＜0.01).The PTEN mRNA level of the siR-996 group,siR-1381 group and siR-1519 group were lower than that of the NC group,the differences were statistically significant(P＜0.01).The PTEN protein content and PTEN mRNA level of siR-996 group were the lowest,the silencing efficiency of PTEN protein content and PTEN mRNA level was higher than that of siR-1381 group and siR-1519 group.Conclusion SiR-996 is a highly efficient siRNA for PTEN gene silencing in mouse primary hepatocytes.
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