I'm looking into IL-6 mRNA expression from circulating leukocytes in my sheep samples.
I'm getting huge variability even before treatment. Is it okay for me to say that the variability is due to different immune background (breed etc - mine are merino cross).
I understand that IL-6 requires a stimulus before it can be expressed. Could it be that stress 2 days prior to treatment (i.e. restraint on table for catheterization for nearly 1/2 hr) could have caused this response?
If anyone knows a link for papers that looked into IL-6 mRNA expression from leukocytes in sheep, I appreciate that!
Are your samples normalised for IL-6 mRNA using a "reference gene/housekeeping gene ?". if not then the issue may be one of different loading or different RT efficiencies. Are you doing absolute i.e real number quantification of the IL-6 using a standard curve from cloned IL-6?. Are your preps standardised i.e the same length of centrifugation over ficoll etc. There may be issues of activation of cells through processing - have you tried looking at cells incubated for different times in TC plates - an initial stimulation response can be reduced after o/n incubation in some instances, in others contact with plastic can be stimulatory.There is always going to be a baseline fluctuation which will usually become smaller with increasing sampling size.
Hi Ian,
Yes, they are normalised to the reference gene, and I am not doing absolute quantification in relevance to the standard curve. I have very limited samples i.e. only 6 per treatment, and small amount of sample to begin with!
It is a baseline study that I wish to observe a trend over time, but definitely further validation is required. I will report the variability due to these limitations.
Thanks Ian.
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