In realtime PCR, my aim is to standardize the stable gene in tomato tissues (control and heat stress). I am facing a problem finding a stable gene expression in both control and heat stress. I tried 18S, Cyclophilin and ubiquitin genes. All are expressing differently among the control vs heat stress. How can I standardize it? How can I use use two endogenous genes? Please help me in this regard.
try HPRT1, as being the gene involved in the generation of nucleotides it should not vary.
You can also try GAPDH as it has been shown to be stable it most tissues.
I would suggest to go and search for what reference genes are commonly used in tomato gene expression. Maybe someone has already had a similar problem. As Chisha suggests, GAPDH is always a good alternative, I could think of Beta2 Microglobulin or Beta-Actin too (At least for mammalian cells work).
I would also suggest to check in your methods that you synthesise cDNA from the same amount of RNA for all your samples. If you can also check the quality of RNA collected from your samples, RNA degradation can influence also your PCR analysis.
Hello,
This paper could help...
Løvdal T1, Lillo C. Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal Biochem. 2009 Apr 15;387(2):238-42. doi: 10.1016/j.ab.2009.01.024. Epub 2009 Jan 24.
Good luck
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