I am looking at cytokine gene expression of IL-10, IL-12p40, IL-17a, Muc-2, and TGF-beta in infant stool samples at 12 weeks of time point. I am looking for a good positive control to use to ensure that my assay will work each time. Can anyone suggest me a positive control? I've tried fMLP stimulated PMNs and unstimulated PBMCs, but neither worked.
I'm not sure about Muc-2 (perhaps an epithelial line stimulated with TNF) but, for the others, some whole blood activated 24h with 100ng/ml LPS and 5µg/mlPHA should do it.
Sorry, in previous answer I should have mentioned it is best to dilute the whole blood in medium (RPMI is good) 1:1 or 1:2
Diana, as Stephen, I can't say about Muc-2, but you should get a signal from the other cytokines. For gene expression, safer to test several time points starting at 2 h of stimulation. Likely IL12p40 starts expression rather early, IL10 mRNA is detectable by 5-6 h stimulation when using 100 ng/ml LPS.
Use a compound (published) that is known to induce their expression.
The best choice for positive controls are synthetic RNA segments that include the relevant sequences of the mRNA of interest. Each positive control is obtained in pure form without any problem of cross-reactivity. The preparation of defined mRNA sequence mixtures is possible. These synthetic RNAs can be in the size range of a few hundred or more than 1000 nucleotides, and they are commercially available in pure form, or in the desired mixtures from the company www.amp-tec.com. Hope this is usefull.
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