Expanding the Diversity of Imaging-Based RNAi Screen Applications Using Cell Spot Microarrays

Microarrays

Juha K. Rantala¹  Sunjong Kwon²  James Korkola² 

[1] Department of Biomedical Engineering and Knight Cancer Institute, Oregon Health and Science University, Portland, OR 97239, USA;

关键词: RNA interference;    high-throughput screening;    cell spot microarrays;    image cytometry;    quantitative immunofluorescence assays;   

DOI  :  10.3390/microarrays2020097

来源: mdpi

【 摘 要 】

Over the past decade, great strides have been made in identifying gene aberrations and deregulated pathways that are associated with specific disease states. These association studies guide experimental studies aimed at identifying the aberrant genes and networks that cause the disease states. This requires functional manipulation of these genes and networks in laboratory models of normal and diseased cells. One approach is to assess molecular and biological responses to high-throughput RNA interference (RNAi)-induced gene knockdown. These responses can be revealed by immunofluorescent staining for a molecular or cellular process of interest and quantified using fluorescence image analysis. These applications are typically performed in multiwell format, but are limited by high reagent costs and long plate processing times. These limitations can be mitigated by analyzing cells grown in cell spot microarray (CSMA) format. CSMAs are produced by growing cells on small (~200 μm diameter) spots with each spot carrying an siRNA with transfection reagent. The spacing between spots is only a few hundred micrometers, thus thousands of cell spots can be arranged on a single cell culture surface. These high-density cell cultures can be immunofluorescently stained with minimal reagent consumption and analyzed quickly using automated fluorescence microscopy platforms. This review covers basic aspects of imaging-based CSMA technology, describes a wide range of immunofluorescence assays that have already been implemented successfully for CSMA screening and suggests future directions for advanced RNAi screening experiments.

【 授权许可】

CC BY   
© 2013 by the authors; licensee MDPI, Basel, Switzerland.