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A
B
Highly Efficient Knockdown Effective High-Throughput Knockdown in 1536-Well Plates
Figure 4 Transfection of siRNA targeted against A the GFP
portion or B the ETAR portion of the fusion protein resulted
in effective knockdown as shown by fluorescence imaging.
Effective silencing was achieved with siRNA amounts
ranging from 0.25 to 2 µg. Control transfections were
carried out with medium only, buffer only, siRNA only, and
RNAiFect Reagent only. Transfections were carried out in
96-well plates.
Ordering Information
Figure 5 Ultra-high–throughput RNAi screening using GFP-22 siRNA in 1536-well plates
resulted in effective knockdown with very low variability across the plate as shown by
membrane fluorescence imaging. Non-silencing control siRNA was also transfected.
Conclusions
■ HPP Grade siRNA Duplexes, cell seeding and liquid handling stations,
and the Opera imaging reader were successfully used for highthroughput
gene silencing of endothelin A.
■ RNAiFect Transfection Reagent provided high transfection efficiencies of
close to 100%, even when cells were transfected in a 1536-well format.
■ The results demonstrate how an assay, such as fluorescent monitoring
of endocytosis with the Opera imaging reader, can be combined with
cost-effective, high-throughput RNAi screening for highly efficient
functional genomics research.
References
1. Chun, M., Lin, H.Y., Henis, Y.I., and Lodish, H.F. (1995) Endothelin-induced Endocytosis
of Cell Surface ETA Receptors. J Biol Chem, 270, 10855.
2. Wang J, Chiou W.J., Gagne G.D., and Wu-Wong J.R. (2000) Internalization of type-A
endothelin receptor. J Cardiovasc Pharmacol, 36(5 Suppl 1):S61.
Product Contents Cat. no.
HPP Grade siRNA (20 nmol)* siRNA purified to >90% –
GFP-22 siRNA (5 nmol) 5 nmol GFP-22 siRNA; for use as a positive (silencing) 1022064
control for green fluorescent protein
Negative Control 5 nmol duplex siRNA with no significant homology to 1022076
siRNA (5 nmol) any known mammalian gene; for use as a non-silencing
control in RNAi experiments
RNAiFect Transfection RNAiFect Reagent and buffer, for up to 170 transfections 301605
Reagent (1 ml) † in 24-well plates; up to 500 transfections in 96-well plates
Find out more about Evotec technologies’ solutions for high-throughput RNAi at www.evotec-technologies.com .
* Available in guaranteed yields of 20 and 40 nmol. Also available in 96-well plates; please inquire.
† Larger sizes available; please inquire.
78 www.qiagen.com QIAGEN News 2004 Issue 3