Epsilon Archive for Student Projects

Abstract

RNA interference is a naturally occurring process of gene silencing that reduces the expression of the gene from which the RNA sequence is derived, when added in a double-stranded form. RNA interference works as defense against viruses and transposons.
The aim of this study was to optimize a protocol for RNA interference in mouse mammary epithelial HC11 cells by using different controls, and finally to silence the drug transporter BCRP to do an uptake-study with the BCRP-substrate mitoxantrone.
The optimization of the protocol was performed with four different lipids, as well as sicontrol tox, negative control and a positive control Cyclophilin B. Sicontrol tox and the negative control were assessed with ATP determination. Real time PCR was performed to check the down-regulation of Cyclophilin B after RNAi treatment of the HC11 cells.
The results of the optimization protocol showed that transfection for 48 h in the presence of lipid nr 1 diluted 1:4 times works best for transfection in HC11 cells. Cyclophilin B was down-regulated about 90 % with this protocol, and BCRP was down-regulated 80 %. The uptake-study with mitoxantrone showed that the BCRP gene silenced cells accumulated the double amount of mitoxantrone as compared to controls.
In conclusion, the obtained results provide a promising model for screening BCRP substrates, which can be used to examine other transporters in mammary epithelial cells, as well.