I am trying to run QPCR on NK cell RNA using SYBR green. I have not been getting any amplification even in the 18S gene which we use as control. While the same primers and SYBR green works well on pancreatic cancer cell lines. Then I ran QPCR from the same cDNA from NK cells using TaqMan probes and got amplification and expected Ct values. What could be causing this variation?
Thanks.
Are the NK cells and the pancreatic cancer cell line from the same species? If your primers are designed for a specific species (e.g. rat, human...), they won't work in a different one... Except if you design them to be general and fit several organisms.
Also, most taqman are species dependent, even if some work in several close species (ex both mice and rat, or human and pig...).
Also, it might be a temperature dependent thing. Try to lower it (0,5 or 1°C at a time), check the melting curve and that you have only one product (agarose gel).
Thanks Sabrina.
Yes both the cells are from the same species i.e human. It's very puzzling why this is happening.
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