Automation Considerations for RNAi Library Formatting and High Throughput Transfection
The ICCB-Longwood Screening Facility at Harvard Medical School is a resource for academic investigators carrying out small molecule and genome-wide siRNA screens. When they began supporting siRNA screens in 2006, they adapted work flows for library management and formatting that they had developed for their small molecule libraries. In this interview, Sean Johnston, Facility Manager, Assay Implementation at ICCB-Longwood HTS Facility, gives advice on making the switch and discusses some of the major differences between workflows for small molecule libraries as opposed to siRNA samples.
Pharma IQ: How difficult was it make the switch to start supporting siRNA screens? What advice can you give labs looking to start this process?
S Johnston: It was fairly straightforward for us to start supporting siRNA screens. The ICCB-Longwood screening facility was first established at the Institute of Chemistry and Cell Biology at Harvard Medical School in 1997 for the purpose of small molecule screening. When we decided to implement genome-scale siRNA screening in 2006, we had the benefit of years of experience with infrastructure, automation, and the logistics associated with effectively managing a large number of projects. Nonetheless, we had to make some major adjustments to our screening workflows to accommodate siRNA screening.
I would advise labs new to the process to carefully consider their projected and desired throughput, their target clientele, and their level of assay support before establishing a strategy for how to proceed. These factors may dictate the selection of equipment, libraries, personnel, and lab space. There are many different ways to run a successful screening center, and it is helpful to look at strategies in place in multiple different facilities before making a decision of how to model your own system.
S Johnston: From the facility perspective, some of the major differences between our small molecule and siRNA workflows are associated with how we handle the libraries. We have different strategies for library formatting, delivery to the assay plates, and storage. From a screener’s perspective, the main difference would likely be the transfection step. Unlike the passive diffusion or active cell transport of small molecules, siRNA requires a vehicle for delivery through the cell membrane, whether via use of a lipid-based reagents or electroporation. We have found that reagent-based transfection is highly sensitive to a number of factors including cell density, cell passage, siRNA and transfection reagent concentration, and reagent lot; variation in any of these conditions may mean the difference between assay success and failure. Controlling these factors over the course of a 3-4 month screening project requires careful consideration.
Pharma IQ: Do you have separate libraries? Are their differences in how you store these two types of compounds?
S Johnston: We have a large variety of chemical and siRNA libraries available at the ICCB-Longwood. The most obvious difference between the two types of libraries is the solvent used. Our chemical collections are dissolved in dimethylsulfoxide (DMSO), while our siRNA libraries are resuspended in an aqueous buffer. These two solvents require different storage methods, as DMSO absorbs atmospheric water which causes the compounds to crash out of solution, while the siRNA buffer can easily evaporate. Precipitation and degradation are our major concerns with chemical libraries, while evaporation and exposure to RNases are main considerations for siRNA collections. We also store these libraries at different concentrations and in different types of labware for a variety of reasons.
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