Endogenous controls (housekeeping) for immunologists - (Mar/15/2006 )
I was wondering whether anyone out there also works in T cell immunology and could help me with choosing an appropriate endogenous control for my real time?
Up until now, I've been using 18S as my endogenous control to quantify the expression of my genes (comparative Ct method) in activated and memory T cells and have used naive T cells as my calibrator.
However, won't activated T cells express more 18S than resting naive T cells?
I've been reading some previous posts in this forum and realise I have a few options such as
1) Trying a bucket load of other endogenous control genes and choosing the best
2) Using two endogenous control genes in that tricky paper by Vandesompele et al
however I was hoping that some of you out there have already been through this and have found a solution.
The other things to mention here are that I'm working with murine T cells, I use sybr so I can't just buy nicely pre validated Taqman assays off the shelf, and the reason we use sybr is because its cheap so option 1 above isn't ideal due to cost and time etc!
Any help would be really appreciated.
18S should be one of the "better" housekeeping genes for your needs. People in my institute also use TBP or Pol2A as housekeeping genes for T cell immunology.
However it really depends how sure you want to be that your results are not artefacts of HKG variation. I would recommend to use 2 or 3 housekeeping genes, at least in figures you want to use for publication. Especially if you use very strong stimuli as e.g. Ionomycin that really push the cells metabolism.
If you work with in vitro systems you can also start with the same amount of cells for RNA isolation and proceed with all samples in parallel. This should result in similar Ct for the HKGs as long as they are stable.
Check thsi paper: http://www.ncbi.nlm.nih.gov/entrez/query.f...l=pubmed_docsum
They compared HKGs in activated T cells (its human, bu still interesting)
Cheers for that RatioPharm.
Also found this paper on human T cells (if you are interested): Bas et al. Scand JI. 59(2004):566-573 and they say 18S is the best.
Just one question about your comment about starting with the same no of cells. I was doing this until I came to my naive v's activated expt and found that I needed 10 fold the number of naive T cells to get enough RNA. Won't this huge difference in no's of cells skew the results?