I wanna detection genes related to NKT cells (receptors), because of these cells are so low in our blood .detection of these genes is so hard and when I try by SYBR® Green Real-Time PCR, My primers amplify and when I use low Con of primers my gene doesn't amplify.
I tested with more amount of cDNA (instead 2 microliters) but in that condition, my genes don't amplify .
what can I do?
what Con of cDNA or RNA do I use for detection of genes?
Digital droplet is indeed a possibility.
If you don't have access to that, an other solution could be a nested pcr: you do a 1st pcr (without reaching the saturation phase to avoid amplifying off targets!) with a set of primers, then a 2nd pcr with primers targeting a region inside your first amplicon. It might need a bit of optimization first, but it is a cheap way to increase the sensitivity of your pcr.
You can try to pool together more cells in the same tube and extract more RNA.
It could also be that the levels of this gene are very low or not even expressed. If you have sufficient amounts of RNA, getting such a result is actually a result in of itself. How many ng of RNA are you inputting for cDNA?
Digital droplet PCR is your best bet. It is significantly more sensitive than RT-PCR as well as properly quantifiable. E.g. You get XX copes/ml of sample.
what if you try to induce higher NK cell production in your model, this will provide you more enriched proportions of NK cells in your culture or cell pool.
Digital droplet is indeed a possibility.
If you don't have access to that, an other solution could be a nested pcr: you do a 1st pcr (without reaching the saturation phase to avoid amplifying off targets!) with a set of primers, then a 2nd pcr with primers targeting a region inside your first amplicon. It might need a bit of optimization first, but it is a cheap way to increase the sensitivity of your pcr.
Thanks,dear Can Kiessling
I wanna detect NKT cells from PBMCs and I shouldn't culture or isolate these cells. Because my aim is evaluate the expression genes of these cells from whole blood. My RNA's ng is 60 micro gram/microliter.
Evelyne Collignon , I optimized my PCR set with control gene(cDNA from cultured cells), but it that condition When I use more primer my gene amplify with a lot of primer dimer and when I diluted my primer my gene doesn't amply and also I try this with more amount of cDNA (instead diluting my primer),But unfortunately doesn't work.(I tried this by q-RT and PCR)
Just pool more blood into the column them. You can request for more blood draws and pool all of them into the same RNA column. However, you need to make sure you don't overload the column. Otherwise your options are more sensitive methods such a digital PCR or label it as not detectable. Again, a negative result can still be labelled a result and can be informative.
Like Evelyne suggested, I would recommend trying nested PCR!
Also, have you already validated your primer set? If you haven't, I would recommend first checking to make sure that your primer set gives you 90-110% amplification efficiency. Once you know that your primers are working at maximum efficiency, then you can play with what you need to do with your cDNA concentrations!
I also work on detecting a gene that has very low expression levels in blood. I've found that using nested PCR not only increases my starting template for easier detection, but also greatly increases the reliability of my assay.
Hope this helps!
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