Overview of the Screening Process
The screening process can be split into a number of discrete steps and organised in two stages between your institute and the SRSF.
(1) Develop your idea and hypothesis:
come up with a theoretical assay you want to test experimentally.
(2) Assay Development - Biology:
develop the reagents in your laboratory working towards 384 well format. It is not necessary, at this time, to have run 384 well tests.
Try some RNAi (dsRNAs) controls in your lab to see if the assay works on suspect targets. If you are satisfied with your results then buy some assay development plates from us. We can give you help about what sort of assays to develop and how you think about testing some hypotheses.
(3) Assay Development - Test Plate:
plates give the user an opportunity to develop a few key aspects of screens.
Primarily, if you use them at the SRSF, you get to determine if the assay works in 384 well format, you also get to use equipment that will save you time and effort during screening (hand-held pipettes are not the answer!).
Using assay development plates you have the chance to use some of our high through-put equipment and begin refining the assay into a tool that you can use with confidence in a screen. If you have acquisition equipment in your department or are able to continue at this stage in your laboratory we can send assay development plates to you.
(4) Decide when you want to start screening:
we then carry out a consultation with our users. Generally this is quite informal, but it's here to help. Your data is presented to members of the SRSF and advice is offered on whether the screen is viable and also how to improve it before commencing.
(5) Complexities of the screen:
depending on the complexity of the screen, users are either first suggested to screen the phosphatome/kinome to develop the process, if it is complex, or go ahead with the genome.
If the user screens the 5 kinome/phosphatome plates first, it gives them an idea of the workstation approach we use. 53 plates with aspiration, washing and dispensing steps can get very labour intensive and hairy at times.
(6) Data analysis:
this should not be taken lightly and can often take you more time than you imagine. For example, if you do a luciferase read-out screen in duplicate you’ll end up with the minimum of 20,000 (size of library with controls) x2 (channels) x2 (replicates) = 80,000 data points.
Most spread-sheet packages are prone to crashing with data sizes this big. We save our data in txt files and process them using the HTS2 package in ‘R’. We can give advice on how to prepare the raw data for processing prior to statistical analysis and what to do with this part of the analysis procedure.