Could you give us a few more details on your experimental setup?

1. What do you mean by ‘appropriate cell type to look at’? Isn’t there a reason why you are looking at spleen/kidney?

2. How are you stimulating your cells?

3. Are you running your products on a gel or are you actually looking at Ct values? What is the Ct value for your house keeping gene? And what Ct value is IL-15 at?

4. Did you try using primers that others have used/reported before?

As far as I know, IL-15 is expressed constitutively and it is not a cytokine that can be induced easily (as opposed to other cytokines). If your house keeping gene is at 10-25 Ct and your IL-15 is >32, chances are you have negligible amounts of IL-15.

1. Try using more concentrated cDNA in your qPCR reaction setup to bring the Ct value for IL-15 within detectable range.

2. Try using primer pairs that others have used and have reported to work.

3. Ensure that your stimulation is indeed supposed to enhance IL-15 transcript levels.

As I mentioned before, details on your objectives and assay setup would help us give recommendations on the cell type/time frame.

Dear Julie,

Thank you very much for your response. Actually I want use mice IL-15 for a cloning purpose as a part of vaccine development experiment. I want to amplify the full mRNA and use for cloning activity. The primers I used in this is not from a published data. I have retrieved IL-15 mRNA information from the NCBI and I designed my own primers. I have notice Tm s for each primer was quite incompatible as Tm for FP is 58 and for RP is 65. However, I was trying to amplify using these primer set in order to get the full construct. I was checking the amplification on a agarose gel, but not by RT-PCR. As you have suggested, I am trying to increase the number of PCR cycles and sea whether I will get the amplification.

Thank you,

Kindly,

Amal.