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Quantification of siRNA Silencing Efficiency,,,Using the LightCycler System

Since the first description of RNA interference (RNAi) as a protection mechanism against invasion of foreign genes appeared a few years ago, RNAi has become a powerful tool for analyzing gene function in mammalian cells. RNAi is mediated by short double-stranded RNAs that are homologous to the suppressed gene. These small interfering RNAs (siRNAs) of two hybridized 21-mer RNA molecules (each containing 19 complementary nucleotides and 3′ terminal overhangs of two nucleotides) mediate sequence-specific mRNA degradation in mammalian cells [1].

To generate siRNA in vitro for transfection into cells, different methods such as chemical synthesis and in vitro transcription are used. Other methods apply vectors to generate siRNA in vivo within the cells. The method of generating siRNA and the siRNA sequence are both important factors for a successful knockdown experiment. Not all chosen siRNA sequences are able to repress gene expression with the same efficiency. A reduction of the mRNA level of > 90% is obtained with the most efficient siRNAs; others have little or no effect. Therefore, three or four siRNAs per target gene are usually tested to find the siRNAs that is most suitable, or in vitro transcribed RNA is digested using DICER enzyme to obtain a mixture of siRNAs. To make sure that an observed silencing effect on the protein level is specific to a used siRNA, the mRNA level of the target gene should also be measured. In addition, the mRNA can also be measured for genes where no specific antibody is available for the gene products. For proteins with a long half-life it is often easier to evaluate the efficiency of siRNAs on the mRNA level, because the knockdown effects are higher.

We have used the LightCycle r System for quantification of the green fluorescent protein (GFP) mRNA level relative to a housekeeping gene mRNA after GFP gene knockdown of a cell with a stably transfected GFP gene. This method was successfully used to show the specificity and silencing efficiency of GFP-specific siRNAs.

Materials and Methods

Cell culture Cells of the human adherent cell line PC3 stably transfected with the gene for the GFP (PC3-GFP) were cultured in RPMI 1640 containing 10% (v/v) fetal calf serum, 2 mM L-glutamine and 750 g/ml neomycin. Two days prior to transfection with siRNA oligonucleotides, 5x104 cells were plated in each well of 24-well plates. The siRNA (Dharmacon) was mixed with diluted transfection reagent and applied to the cells in a 100 nM concentration in medium without serum (Opti-MEM). After 4 hours of transfection, fetal calf serum was added to 10% (v/v) final concentration.

GFP assay GFP concentration was measured in 100 l lysate with a spectrofluorophotometer RF-5301 (wavelength: EX: 395 nm, EM: 508 nm).

Isolation of mRNA The mRNA was isolated with the MagNA Pure LC Instrument using the MagNa Pure LC mRNA Isolation Kit I according to the kit protocol for cultured cells and the MagNA Pure protocol mRNA I cells. Elution volume was 50 l.

cDNA synthesis Using the 1st Strand cDNA Synthesis Kit for RT-PCR with random primers according to the kit protocol, 5 l mRNA of each sample was transcribed into cDNA. The cDNA was diluted tenfold in PCR-grade water and quantified with the LightCycler Instrument.

Quantitative real-time PCR For the GFP and housekeeping gene amplifica tion, the LightCycler FastStart DNA Master SYBR Green I Kit and LightCycler FastStart DNA Master Hybridization Probes Kit were used according to instructions: 0.5 M of each primer, 0.2 M HybProbe Probes and 4 mM MgCl2 were applied. The GFP-specific primers and HybProbe Probes were designed by the LightCycler Probe Design Software 1.0. Amplification was performed under the following conditions: a pre-incubation step for 10 minutes at 95C followed by 45 cycles of 10 seconds at 95C, 10 seconds at 60C, and 15 seconds at 72C (temperature ramp was 20C/seconds).

Results The human cell line PC3-GFP stably expresses GFP and shows a strong fluorescence signal (Figure 1A); 72 hours after transfection with chemically synthesized GFP-specific siRNA (GFP siRNA), the fluorescence signal was significantly reduced (Figure 1B).



The GFP knockdown was measured on the protein level. GFP expression was reduced by 53% (ng GFP/g total Protein) in PC3-GFP cells transfected with GFP siRNA compared with cells transfected with luciferase siRNA (Figure 2), which showed no GFP knockdown.



The LightCycler System is ideally suited for relative quantification of target mRNA levels in relation to reference gene mRNA levels. To determine the specificity of the used GFP siRNA on the mRNA level, a quantitative real-time PCR was performed using the LightCycler System.

In a first experiment, the different housekeeping gene expression levels in PC3-GFP cells were determined. The most suitable housekeeping gene was selected as a reference for relative quantification. GFP amplification and detection was performed with the LightCycler FastStart DNA Master SYBR Green I Kit, whereas the amplification and detection of the housekeeping genes were carried out using the LightCycler h-Housekeeping Gene Selection Set and the LightCycler FastStart DNA Master Hybridization Probes Kit. The data from the housekeeping genes were used to normalize the GFP results (Figure 3). Of the five tested housekeeping genes (β2M, G6PDH, ALAS, HPRT and PBGD) the expression level of β2Microglobulin (β2M) was closest to the expression level of GFP (Figure 3a). Therefore it was selected as reference for relative quantification in the second experiment. The down-regulation of GFP mRNA was in the same range, independent of the housekeeping gene used as a reference for relative quantification. GFP mRNA was down-regulated by 81%89% by GFP siRNA in comparison to luciferase siRNA, which showed no down-regulation (Figures 3a, b).



In a second experiment, the GFP knockdown was detected using HybProbe Probes. The resulting quantification data were normalized with the β2M quantification data. The amplification of the GFP cDNA showed a strong reduction of GFP mRNA in PC3-GFP cells transfected with GFP siRNA compared to PC3-GFP cells transfected with luciferase siRNA (Figure 4b). GFP mRNA was down-regulated by 85% with GFP siRNA compared with luciferase siRNA. In contrast, the expression level of the housekeeping gene in PC3-GFP cells transfected with luciferase siRNA, or with GFP siRNA was nearly identical (Figure 4a).



Conclusion

To confirm the specificity of a down-regulation caused by siRNA, it is necessary to show that the down-regulation on the protein level can also be detected on the mRNA level. Especially for very stable proteins like GFP it is often difficult to see a high knockdown on protein level. Using the LightCycler System, we could show that the GFP siRNA could effectively reduce GFP mRNA by 85%, whereas the reduction on protein level is only 53%. For detection of the target GFP mRNA, we used either SYBR Green I or HybProbe Probes. With both methods we got nearly identical results.

The LightCycler System offers an easy and fast method to confirm that a used siRNA can specifically knockdown target mRNA.


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