I am culturing bEnd.3 cells (mouse brain endothelial cells). I have noticed my Ct values for the same cDNA input amount increase. For example, GAPDH went from Ct 13 to 20 when using cells from passage 15 versus 19. My other genes also increased, so much that one is undetectable at certain dilutions it was previously detectable at. I have heard that this is typical but how do I know what passage is best for qPCR? Is there a general rule of thumb for primary cells?
I have already checked cDNA synthesis, primers, and RNA isolation. The only changing variable seems to be the passage number.
Any resources/literature on this topic would be helpful. Thanks!
Hi Kassondra,
Primary cells are well known difficult to culture. You need to add many growth factors to maintain their viability. Therefore, it is reasonable to get increased Ct values when using the same amount of cDNAs. It means the cell state becomes poorer and poorer, and goes to death finally. Poor cell state will definitely affect RNA/cDNA quality. Have you checked the cell viability or cell state before collecting RNA?
On the other hand, we usually make the Ct values between 17-30, thus you can choose the corresponding passage number to make most of your data within this range.
Hope this information can help you.
You are saying primary cells but they are not primary cells.
Primary cells are isolated from an animal and are grown in vitro or analysed in situ.
If you actually isolated from a mouse brain endothelial celsl with surgery and grew it in cell culture, then that would be a "primary" cell.
It is known that morphologically "primary" cells deteriorate over days and passages so a change in gene expression would be expected. However, ATCC states that your cells are transformed cells.
You should perhaps consult ATCC or experts that have used these cell lines. Perhaps it is not an unusual phenomena.
When I was growing HL-1 cardiomyocytes, we avoided using >10-20 passages. We also performed reference gene stability assessment to avoid such issues as you were mentioning.
Have you checked the status of your cells for mycoplasma (a known effector of gene expression)?
Thanks for the great information and suggestions! Thanks for pointing out the primary vs. transformed cells. I had only noticed that they were isolated from 6 week old mice. I have not checked for mycoplasma but will look into it. Thanks for reassuring me that passage number could influence this greatly. I will also contact ATCC.
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