I'm trying to study the function of a gene on cancer cells and would like to study the gene knockdown/knockout model. I found various ways to do study the model but have difficulty choosing the appropriate methods.
Which would be the best methods among the followings and their pros and cons?
-CRISPR-mediated gene knockdown
-RNA-based interference(siRNA or shRNA)
-Drug antagonist/neutralizers
If I were to used the drug antagonist/neutralizers, how should I determine the appropriate drug dosage? eg. MTS assay, etc.
The easiest and fastest way would be using siRNA if you only need to see a transient effect. CRISPR KO/KD will be long lasting assuming the cells survive. If it is an essential gene, it would be a problem.
Don't ask random folks on the internet to answer your homework for you.
If this happens to be for your actual research, then go read the literature.
Dear Htoo Shwe Eain, If you work with cell lines, siRNA-based knockdown would be better to perform. If you work with mice and with micro-injection facilities CRISPR based KO can be performed. We have experience with siRNA-based KD in cell cultures. There you need to design the experiment from primer designing. For detailed protocol read this chapter “The Use of Double-Stranded RNA to Knock Down Specific Gene Activity" (https://link.springer.com/protocol/10.1385%2F1-59259-755-6%3A129)
siRNA delivered with transfection reagent such as RNAiMax. if prolonged effect is required- you can do shRNA
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