I have tried to separate a direct coculture of MSCs (mesenchymal stromal cells) and macrophages to do bulk RNA seq on macrophages, as I want to find out how MSCs change the genetic expression on macrophages. I have tried different methods to separate the coculture as much possible, but I can only manage to retrieve a cell population with 95% macrophages, and 5% MSCs still present.
Therefore, I want to know if anyone has experience with analyzing data when the population is not completely pure with one cell type and how do I handle such data?
Is it wise to proceed with bulk RNA seq when 5% of my cells are still MSCs, well aware that the expressed genes observed could come from the 5% MSCs?
A general guideline, a contamination level of less than 10% is often considered acceptable for bulk RNA sequencing.
Dhananjay Kumar thank you for your answer, much appreciated! But do you mind me asking if it's based on your own experiences or do you have any references stating the same?
Dear Kian,
have you tried improve your purity by FACS? It´s fairly easy to choose markers to distinguish MSC & macrophages and sort highly pure populations.
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