For my project, I need to isolate Th17 cells out of skin and check gene expression via qPCR. Does anyone have some experience with this or knows of an alternative way to address this? All suggestions are welcome. Thanks!
Hi Marlies,
could your give some more details on your scientific question? Is it human or mouse skin? Do you have specific genes in mind?
If there are antibodies available, flow cytometry or histology might be interesting alternatives for you to directly check the protein level.
If you have many genes, you might directly want to do RNA Seq or microarray analysis. I have obtained microarrays of human blood Th17 cells sorted with the cytokine secretion assay for IL-17A expression (Manz, PNAS, 1995). Although rather rare (2-3% of memory phenotype Th cells), I could isolate sufficient numbers of cells from the blood (100.000, ca. 100ng RNA), however this might be difficult for skin. Now, there are microarray kits out there that are supposed to work with as little as 0.5ng corresponding to 500-1000 Th17 cells. If you go for qRT-PCR, you might need around 3-5ng as other people have stated here on RG.
Alternatively to the cytokine secretion assay, you could use chemokine receptor expression like CCR6, CXCR3 etc. to distinguish the different subsets (e.g. Sallusto, EJI, 2009).
Good luck!
Jakob
Thanks for your input Jakob and Nicole.
I have to isolate Th17 cells out of mouse skin and check the expression of only one gene. Unfortunately, detection of protein levels is rather difficult, so I think stainings will be difficult to optimize.
Does it definitely need to be Th17 cells? Maybe the gene is anyway exclusively expressed by Th17 and bulk CD4 cells would do the job?
Otherwise maybe ROR-gt-GFP mice could be an option to isolate Th17 cells?
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