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subsequently dissociates from endothelin-1 and is either recycled back to the cell surface (2) or
degraded. High-throughput RNAi techniques can be combined with monitoring of endocytosis
using a fluorescent marker, for the identification of genes involved in the internalization process
(Figure 2). Similarly, the combination of RNAi techniques with any appropriate assay can be used
for screening large numbers of genes potentially involved in cellular processes.
Materials and methods
HPP (High-Performance Purity) Grade siRNA duplexes from QIAGEN were targeted against either
the ETAR or the GFP portion of the ETAR–GFP fusion protein. The target sequences of the siRNA
duplexes were: ETAR siRNA 5'-AACGCGCTGATAGCCAGTCTT-3', GFP-22 siRNA
5'-CGGCAAGCTGACCCTGAAGTTCAT-3'. The sequence of the non-silencing siRNA duplex was
5'-AATTCTCCGAACGTGTCACGT-3'. siRNA transfections were performed in 96-well or 1536-well
microplates using RNAiFect Transfection Reagent. For transfections in 96-well plates 4 x 103 CHO
cells were seeded per well and allowed to adhere overnight. Cells were transfected with 0.25 µg,
0.5 µg, 0.75 µg, 1 µg, 1.5 µg, or 2 µg siRNA and after 48 hours plates were analyzed on the
Opera imaging reader. Experiments in 1536-well plates were carried out using Evotec
Technologies’ ultra-high–throughput cell seeding and liquid handling stations. Three hundred CHO
cells were seeded per well and allowed to adhere overnight. Cells were transfected with 0.05 µg
GFP-22 siRNA or non-silencing control siRNA. Cells were analyzed 48 hours after transfection.
Plates were read on the Opera imaging reader and the degree of receptor silencing was quantified
by an Opera inherent image analysis algorithm that focuses on membrane fluorescence intensity.
Results
Fluorescence Imaging Shows Effective Gene Silencing
Untransfected 0.25 µg siRNA
1.0 µg siRNA
The gene silencing efficiency of siRNA duplexes targeted against the ETAR–GFP fusion protein was
tested by transfection of ETAR or GFP-22 siRNA duplexes into cells in 96-well plates. The transfection
efficiency was close to 100%. Effective knockdown of the fusion protein was achieved with as little
as 0.25 µg siRNA per well irrespective of whether the ETAR or the GFP-22 siRNA was used
(Figures 3 and 4).
For experiments in 1536-well plates, cells were transfected with GFP-22 siRNA or non-silencing
control siRNA. High-throughput transfection and gene silencing in 1536-well plates was highly
efficient and reproducible, with very little variability across the plate and only small variations in
single-well data (Figure 5). These results clearly demonstrate that RNAi can be used for automated
large-scale gene silencing applications such as screening siRNA libraries for drug discovery or
pathway analysis.
Figure 3 Transfection of
siRNA targeted against the
GFP portion of the fusion
protein resulted in effective
knockdown as shown by
fluorescence imaging.
Effective silencing was
achieved with as little as
0.25 µg siRNA.
QIAGEN News 2004 Issue 3 www.qiagen.com 77