I have three breast cancer cell lines that are being treated with siRNA targeting a specific gene that is known to influence tumour growth. I've been told to leave them for 24hrs after treatment before replating the cells as single cell suspensions for a colony formation assay. What's the reason for waiting 24hrs?
Also would there be any benefit in measuring the total surface area of tumour colonies on the plate as well as counting the number of colonies formed? Would this provide any different information?
Hi,
What I am understanding, it is
1. 24hr could be the time period for siRNA to silence your target gene. So, once that gene gets targeted, which is the objective, then plating for assay will give your answer that what effect has happened on silencing.
2. Yes, the measuring surface area of colonies has its own importance. Cells will form colonies, and these colonies somehow resemble cancer stem cells feature as mammosphere or spheroid or cancer stem-like cells (CSCs). Such colonies or spheroids gives an idea about anchorage-independent growth and tumor recurrence capacity. Size matters for in-vitro culture since particular size colonies are being used for testing.
Pardon me, if my understanding is wrong.
Regards
Saurabh
Hi Jasmine
Different targets need different time to be inhibited by siRNA machinery. Normally this time ranges from 24h to 72h. If this siRNA was already used in this cell line by others from your lab, ok. If this is the first time, you could run a small test treating cells for 24h, 48h and 27h. Then extract check the expression of the protein by western blotting to see when it loses its expression before going for a full experiment.
Regarding the cell suspension colony formation assay, measuring the total number of colonies and their surface is both important and convey different information. By counting the total number of colonies, you are analyzing how well the cells can survive without anchorage, which tells you they are resistant to anoikis. On the other hand, by measuring the area of the colonies you get to know not only if the cells are able to survive but also to measure their proliferation capacity without anchorage.
However, as you are using siRNA and the cell suspension colony formation assay takes around 10 days, you may not see any difference in the end, or dilute this difference, because the protein will be expressed again during the assay. siRNA transfection knockdown lasts for a maximum of 4-5 days (and depends on cells and conditions). For this type of long assays, you want silence genes for longer times, try transfecting with a shRNA instead.
Best wishes,
Priscila
The siRNA usually take 24hr or longer (may vary with cell types and siRNA) to start showing a phenotype and can be checked using western blot. The loss of protein is a good time to start the proliferation experiment so the role of that protein is evident.
Counting the spheres shows how many anchorage-independent cells with the potential to proliferate is there in a population (sphere initiating cell) and the size will help determine the proliferation potential of those cells.
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