A 1,536-well ultra-high-throughput siRNA screen to identify regulators of the Wnt/beta-catenin pathway.
High-throughput siRNA screens are now widely used for identifying novel drug targets and mapping disease pathways. Despite their popularity, there remain challenges related to data variability, primarily due to measurement errors, biological variance, uneven transfection efficiency, the efficacy of siRNA sequences, or off-target effects, and consequent high false discovery rates. Data variability can be reduced if siRNA screens are performed in replicate. Running a large-scale siRNA screen in replicate is difficult, however, because of the technical challenges related to automating complicated steps of siRNA transfection, often with multiplexed assay readouts, and controlling environmental humidity during long incubation periods. Small-molecule screens have greatly benefited in the past decade from assay miniaturization to high-density plates such that 1,536-well nanoplate screenings are now a routine process, allowing fast, efficient, and affordable operations without compromising underlying biology or important assay characteristics. Here, we describe the development of a 1,536-well nanoplate siRNA transfection protocol that utilizes the instruments commonly found in small-molecule high throughput screening laboratories. This protocol was then successfully demonstrated in a triplicate large-scale siRNA screen for the identification of regulators of the Wnt/beta-catenin pathway.
Chung
Namjin
N
Department of Automated Biotechnology, Merck & Co., North Wales, Pennsylvania, USA. namjin.chung@bms.com
Marine
Shane
S
Smith
Emily A
EA
Liehr
Robert
R
Smith
S Todd
ST
Locco
Louis
L
Hudak
Edward
E
Kreamer
Anthony
A
Rush
Alison
A
Roberts
Brian
B
Major
Michael B
MB
Moon
Randall T
RT
Arthur
William
W
Cleary
Michele
M
Strulovici
Berta
B
Ferrer
Marc
M
D000465:Algorithms; D000818:Animals; D002478:Cells, Cultured; D003627:Data Interpretation, Statistical; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation*; D015723:Gene Library; D006801:Humans; D008904:Miniaturization; D034741:RNA, Small Interfering / Q000494:pharmacology* / Q000627:therapeutic use; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured; D051153:Wnt Proteins / Q000235:genetics / Q000502:physiology*; D051176:beta Catenin / Q000235:genetics / Q000502:physiology*
D034741:RNA, Small Interfering; D051153:Wnt Proteins; D051176:beta Catenin