Quantitative optimization of reverse transfection conditions for 384-well siRNA library screening.
A necessary step in all small interfering RNA (siRNA) library screens is introduction of the siRNA into cells. We describe the use of a commercially available glyceraldehyde 3-phosphate dehydrogenase enzymatic assay that is capable of simultaneously assessing the efficiency of siRNA delivery into cells and the lipid toxicity. This assay has been modified to work in 384-well plates using reverse transfection. The assay is fast, inexpensive, and quantitative. Conditions identified as optimal using this technique have been employed successfully in library screens.
LaPan
Peter
P
Section of Biologic Research, Department of Biological Technologies, Wyeth Research, Cambridge, MA 02140, USA. plapan@wyeth.com
Zhang
Jing
J
Pan
Jing
J
Haney
Steve
S
D001483:Base Sequence; D002452:Cell Count; D045744:Cell Line, Tumor; D003627:Data Interpretation, Statistical; D015723:Gene Library; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D008055:Lipids / Q000633:toxicity; D034741:RNA, Small Interfering / Q000494:pharmacology*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods*
D007202:Indicators and Reagents; D008055:Lipids; D034741:RNA, Small Interfering; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases