Caged substrates applied to high content screening: an introduction with an eye to the future.
The use of photoremovable protecting groups in biology affords the end user high temporal, spatial, and concentration control of reagents and substrates. High content screening and other large-scale biology applications would benefit greatly from these advantages. Herein, we report progress in this field by highlighting the recent development of controllable siRNA (csiRNA), which is a dormant siRNA that can be activated using 365 nm light. Two different experimental designs are described to highlight the temporal and concentration variables that can be controlled. First, the RNAi process is activated at two timepoints, 24- and 48-h post-transfection, to demonstrate that the action of csiRNA does not begin until activated. Second, increasing light dosage exposure to cells transfected with csiRNA that controls the concentration of active siRNA molecules. All experiments are conducted in a 96-well format with light delivered through the UCOM device.
Conrad
Peter G
PG
Genospectra, Inc., Fremont, CA, USA.
Chavli
Rajesh V
RV
Givens
Richard S
RS
D000199:Actins / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000172:deficiency / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008027:Light; D034741:RNA, Small Interfering / Q000378:metabolism / Q000528:radiation effects; D013997:Time Factors; D046888:Tissue Array Analysis / Q000295:instrumentation / Q000379:methods* / Q000639:trends*; D014162:Transfection
D000199:Actins; D007202:Indicators and Reagents; D034741:RNA, Small Interfering; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)