We are probing primers designed for TNF-alpha, IL-6, IL-17, IL-23, IL-10 and IL1b, in order to determine expression levels of these cytokines in heart of mice infected with Trypanosoma cruzi. The paper that we got the primers sequences reported a PCR product between 100-300 bp, but we obtained pcr products of 50 bp in heart. We did a positive control with spleen of infected mice, but in that case the products are quite different, higher to 300 bp. If anyone has experience in this topic and can help me, I would be very thankful.
It seems that on agarose gel electrophoresis, you have observed 50 bp product. Confirm it whether it is 50 bp product or it appears to be 50 bp, may be due to primer dimer. otherwise you can confirm it through sequencing of purified gel extracted PCR product.
I agree with Dr Manuja (above); the 50bp product is almost certainly a primer dimer. To help you, you'll need to describe the reactions in question, in detail...
I'd always encourage you to design your own primers rather than take those from a paper, especially if it's more than a couple of years old. The reason is that PCR has advanced technically so far in such a short space of time. Also so many things can affect a reaction that you really need to work it up using your own tubes, reagents, machine... Basically it's easier working it from scratch than trying to troubleshoot someone else's.
You have 6 qRT-PCR reactions, I assume (TNF, IL-1b, IL23p19, IL-10 and IL-6). First, decide on the annealing temperature of the primers that you want for all the reactions, the product length range and the reaction efficiency acceptable to you. It's perfectly possible to design 6 (or sixty) qRT-PCRs with the same conditions; unless they have the same reaction efficiencies, you can't cleanly compare their relative levels. Then, make sure your primers are bedded in different exons. Have one primer wholly within an exon and the other overlapping an exon-exon junction by three or four nucleotides (not more). Be SURE that those 3 or 4 nucleotides do not match the intronic sequence adjacent to the exon where the bulk of the primer is located otherwise you may get DNA amplification. Now, prepare a standard source or cDNA that contains all your targets - splenocytes activated with poly-IC and LPS (together) will be good for the cytokines you're suggesting. Lastly, you're now able to begin testing primer sets and working out the reaction efficiencies in order that you can begin your experiment. Of course, your housekeeping genes (two is preferable) have to be designed the same way. Avoid intronless genes as housekeepers. Good luck!!
G
ps - my answer assumes you have access to a real-time apparatus. If you do not, then it is very difficult to get reliable quantitative data, even relative quantitative data.
As the collegues above, I also think that products in agarose gell, wich you detected are due to the primers. I have a great experience of cytokine gene expression and do not expect transcription of these genes in heart cells. Cytokine genes usually transcribed in immunocompetent and epithelial cells after appropriate activation. Moreover, for accurate detection of transcription you must use qRT-PCR assay.
I totally agree with the above. When you have lower expression in tissue, you tend to get more chance for primer dimmer.
Just to add.. Dr Stanislova is correct that cardiac myocytes would not normally be strong cytokine producers, but under inflammatory or infection conditions, it is surprising what non-immune cells will make (TNF, IL-1 and IL-6 in particular), and of course there may well be infiltrating cells present. What may be useful as a follow up study, is to conduct immunohistochemistry on the infected hearts vrs non-infected ones (or treatment vrs no treatment), staining for the cytokines you find by qRT-PCR. A good pathologist will be able to help you identify the infiltrating cells (neutrophils, lymphocytes, macrophages and so on) and this will guyde your selection of cell-surface markers with which to counterstain, thereby identfying not only the cytokine, but the cell making it, and its location within the tissue.
Good luck!
G
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