A 96-well surrogate survival assay coupled with a special short interfering RNA vector for assessing cancer gene targets with enhanced signal/noise ratio and its utility in HTS for cancer therapeutic targets.
Transient transfection of short interfering RNAs to inactivate cancer therapeutic genes in cancer cells is an important method to induce therapeutic phenotypes (cell apoptosis, growth arrest, etc.) for cancer target validation. These phenotypes can be initially assessed by cell survival via colorimetric/fluorescence readings, e.g., alamarBlue (Trek Diagnostic Systems, Cleveland, OH) and WST-1. However, intrinsic problems exist for transient transfection-varying toxicity, inconsistent transfection efficiency, as well as other cell-specific determinants-which contribute to a low signal:noise ratio of the assays, rendering of the assay ineffective particularly when applied in high-throughput screening (HTS) multiplexed for different cells. This report describes a method using reporter as a "normalized surrogate" for the conventional survival readout in a 96-well format. In this approach, only the transfected surviving cells produce reporter activities, and many variables associated with transient transfection are excluded. A constitutively expressed reporter gene (luciferase or LacZ) expression cassette is co-transfected into cells along with a specially designed RNA interference (RNAi) vector (or a transgene for that matter). The reporter activity in either liquid cultures or in soft agar cultures in 96-well formats is then quantitated in situ. The RNAi vector construction is simplified so that it can be adapted to a 96-well format. Our data demonstrated that the relative reporter readings for survival are independent of both transfection efficiency and cellular toxicity. The signal:noise ratio is markedly increased, particularly for cells with low transfection efficiency. The assay is versatile and robust and can be applied in multiplexed HTS for cancer target identification and validation.
Yu
De-Hua
DH
Immusol, Inc., San Diego, CA 92121, USA.
Chatterton
Jon E
JE
Bliesath
Joshua R
JR
Sundaram
Roshni
R
Ke
Ning
N
Nguy
Vivian
V
Meyhack
Bernd
B
Wong-Staal
Flossie
F
Li
Qi-Xiang
QX
D001483:Base Sequence; D002470:Cell Survival; D004353:Drug Evaluation, Preclinical / Q000379:methods; D017930:Genes, Reporter; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010078:Oxazines; D011519:Proto-Oncogenes / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D014966:Xanthenes; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism
C494826:BAX protein, human; D007202:Indicators and Reagents; D010078:Oxazines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D014966:Xanthenes; D051028:bcl-2-Associated X Protein; C005843:resazurin; D008156:Luciferases