Housekeeping gene for Real Time PCR when comparing expression of genes across di - (Mar/30/2015 )

Hi everyone,

Just wondering if anyone could help! I am trying to compare the expression of various genes across a panel of breast cancer cell lines using Real-Time PCR however I am having trouble finding an appropriate house keeping gene. I have tried ones such as Ubiquitin C, Beta-Actin and GAPDH, however I am finding differences in expression between the cell lines. For example, UBC expression appears to be more highly expressed in some cell lines compared to others so when I am normalizing my gene of interest to UBC, it looks like my gene of interest is increased even thigh Ct values are similar.

Can anyone suggest a gene that stays relatively stable in different cell lines? Or is it not possible to accurately measure gene expression at all in different cell lines?

Any help would be much appreciated :)

Amy

There are not any genes that are stable every time. But usually it's possible to find genes that are reasonably stable in your specific conditions.

But cancer cell are just so deregulated in general, and finding such is very difficult. You need to test and try to find a stable one.

Beta-actin is problematic just by the fact it has pseudogenes but everyone still use it, in cancer it probably isn't stable anyway. GAPDH is not suitable for any cancer cell line since it is a gene of glycolytic pathway, that is often activated in cancer cells by HIF pathway (unfortunatelly many of common reference genes are in glycolytic pathway).

Try to look here:

http://www.protocol-online.org/forums/topic/32220-reference-genes-selection-and-validaton-for-qpcr/