RNA interference : optimizing a protocol for HC11 cells
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 protoc...
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| Formato: | L3 |
| Lenguaje: | Inglés |
| Publicado: |
SLU/Dept. of Biomedical Sciences and Veterinary Public Health (until 231231)
2007
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| Materias: |
| _version_ | 1855571854618525696 |
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| author | Danielsson, Louise |
| author_browse | Danielsson, Louise |
| author_facet | Danielsson, Louise |
| author_sort | Danielsson, Louise |
| collection | Epsilon Archive for Student Projects |
| description | 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. |
| format | L3 |
| id | RepoSLU11344 |
| institution | Swedish University of Agricultural Sciences |
| language | Inglés |
| publishDate | 2007 |
| publishDateSort | 2007 |
| publisher | SLU/Dept. of Biomedical Sciences and Veterinary Public Health (until 231231) |
| publisherStr | SLU/Dept. of Biomedical Sciences and Veterinary Public Health (until 231231) |
| record_format | eprints |
| spelling | RepoSLU113442017-10-09T09:50:55Z RNA interference : optimizing a protocol for HC11 cells Danielsson, Louise RNA Interference HC11 cells 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. SLU/Dept. of Biomedical Sciences and Veterinary Public Health (until 231231) 2007 L3 eng https://stud.epsilon.slu.se/11344/ |
| spellingShingle | RNA Interference HC11 cells Danielsson, Louise RNA interference : optimizing a protocol for HC11 cells |
| title | RNA interference : optimizing a protocol for HC11 cells |
| title_full | RNA interference : optimizing a protocol for HC11 cells |
| title_fullStr | RNA interference : optimizing a protocol for HC11 cells |
| title_full_unstemmed | RNA interference : optimizing a protocol for HC11 cells |
| title_short | RNA interference : optimizing a protocol for HC11 cells |
| title_sort | rna interference : optimizing a protocol for hc11 cells |
| topic | RNA Interference HC11 cells |