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Silencing of Human Choline Kinase Expression

 

Experimental Design

In vitro chemically synthesized RNAi was utilized to knock down expression of the gene for human choline kinase (ChoK). This enzyme has been proposed as a target for antineoplastic drug therapy. Silencing of ChoK by RNAi creates a model situation for studying the consequences of reduced enzyme activity in vivo.

Human MDA-MB-231 breast carcinoma cells were transfected with two different siRNAs (ChoK siRNA 01 and 02) directed against human ChoK mRNA. Cells were lysed 72 hours after transfection, and total RNA was isolated using the High Pure RNA Isolation Kit including DNase digestion. Equal amounts of total RNA were reverse transcribed using the Transcriptor First Strand cDNA Synthesis Kit with anchored oligo(dT)18 primers. Samples were analyzed by real-time PCR after cDNA synthesis using the LightCycler® FastStart DNA MasterPLUS HybProbe. For simultaneous quantification of the housekeeping gene 5-aminolevulinate synthase (ALAS), the detection mix from the LightCycler® h-ALAS Housekeeping Gene Set was added to the same reaction mix. Cross-talk between fluorescence channels was corrected using a color-compensation file. ChoK cDNA levels were normalized to those of the housekeeping gene ALAS using the LightCycler® Relative Quantification Software.

Figure 1: Analysis of reverse transcribed total RNA using the LightCycler® 2.0 System. 5-aminolevulinate synthase (ALAS) cDNA (a) and choline kinase cDNA (b) from samples transfected with siRNAs against ChoK (ChoK siRNA 01 and 02) or control siRNA against luciferase, mock-transfected (transfection agent alone) or untreated (Opti-MEM alone) cells, were amplified on the LightCycler® 2.0 Instrument using ALAS-specific primers and Hybridization Probes labeled with LC Red 640, and ChoK-specific primers and Hybridization Probes labeled with LC Red 705. Each transfection was performed in duplicates.

Figure 2: Knockdown of choline kinase expression. Expression of ChoK in siRNA-transfected samples and controls was normalized to the expression of the housekeeping gene ALAS. Mean expression levels of duplicates relative to the mean of all negative controls are shown.



Results

All samples displayed very similar levels of ALAS, which confirmed the suitability of this gene as a housekeeping control in the transfection experiments (Figure 1a). Both siRNAs against ChoK were able to reduce ChoK mRNA to approximately 13% of control levels (mean of luciferase siRNA, mock treatment, and no treatment), whereas neither luciferase siRNA nor transfection agent alone had an influence on ChoK mRNA levels (Figures 1b and 2). These results demonstrate that transfection of MDA-MB-231 cells has worked efficiently and both ChoK-specific siRNAs are functional and specific.

For further information on the LightCycler® System and its applications, click here.