I have found that a specific treatment influences the Ct value of both the reference gene (Actin and Ubiquitin) in RT qPCR experiment. Is it possible to analyze such data?
If the expressions of your housekeeping genes are modified, then they are not suitable to be used as reference genes in your experiment. I would first recalculate my RNA/cDNA concentration to validate whether it was really the treatment that caused an alteration of actin and ubiquitin expression in treated groups or if it was a concentration error prior to carrying out the qPCR.
If the concentrations are still the same and have been normalized, perhaps you might want to look into other housekeeping genes (e.g. GAPDH or RPL13A)
- If your reference gene is affected by treatment, you absolutely must select an alternative ref gene !!
- As mentioned by Andrew you could try gapDH and RPL13A
- You could add b tubulin and B2 micro globulin to that list
I agree. You do need a valid loading control. If you can't use another gene (reference gene), then you will need to make sure to have equal loading or to correct for different loading levels by other means. For instance, if you can control the number of cells used to isolate RNA, you can add a spike-in control (some synthetic RNA) and use this.
If one reference gene expression is altered by treatment, go with other housekeeping gene. Some physiological factors, such as hypoxia and diabetes, increase GAPDH expression in certain cell types.
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