Hello,
I have been trying to find studies that have FACS sorted E.coli in bulk for RNA extraction and RNA-seq. Maybe I am bad at lit-searching, but I have been surprised to not find a clear answer.
I need at least 100ng of total RNA for rRNA removal and library prep.
My assumption was that I would need 100 million cells, which is based on the estimate that a single Ecoli cell has 2pg of mRNA.
However, sorting 100 million cells is not a reasonable number to sort as I have been told, because the resulting sample volume would be enormous (think >500mL) and would take several hours.
Is my assumption wrong about how many cells are needed? Could we achieve 100ng with far fewer cells?
If you could provide your personal experience or a paper that gives this cell number, I would be extremely grateful!
Best,
Morgan
I don't know what FACS means and your point but I am sure you got it completely wrong.
If you are going use kit for extraction, read the protocol line by line.
I've never ever heard that you mentioned about amount of RNA per cell. It depends on many factors which most important one is using bacteria in log phase for extraction.
I'm sure my answer is awful but I can't do better with your information.
Regards.
This strongly depends on number of inserts/cell, transformation efficacy and reproduction-affecting conditions.
This means that you have to check the amounts needed for each expreiement.
Alireza and Andreas,
I ended up answering my own question. As mentioned above, I needed 100 ng of total RNA, and I had calculated the amount of mRNA per a cell, so the cell number that was estimated is too high. I should only need to sort 2 million cells, instead of 100 million to achieve 200 ng of total RNA.
Thanks for your input!
Morgan
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