CDKN2A gene expression in pancreatic cancer cells is between Ct 36-38 at 50 and 100ng (Enzyme Activation 95oC, Denaturation 95oC, Annealing/Extension 60oC). What could be the reason?
Hi Zeynep,
it is hard to answer your question with the very little information you provide.
1. What do you mean with 50 and 100 ng? Was this the amount of RNA you used for cDNA synthesis? Or did you use DNA directly as a template?
If you have used RNA for cDNA-synthesis you should check your RNA on a gel to see if the RNA is still intact or if you see signs of degradation (before cDNA synthesis). The same is true if you use DNA directly
2. What is the CT value of your positive control (I hope you have a positive control)
3. What is the CT value of your negative (no template) control - you should always include a negative control.
4. Which system did you use for your PCR? SybrGreen, Taqman?
Without further information it is not really possible to answer your question - however I would consider your CT values as really high meaning that your total template concentration is really low (for this you would need a positive control and also the negative control - if you get the same CT values from your negative control as from your sample your signal is most likely background noise).
If you get a good signal from your positive control your template is likely intact and your high CT values mean either a low expression of your gene of interest, however it could also indicate problems with the PCR itself (look at the melting curve). Make sure that you have a good working PCR and a good template in the first place.
KR Dörthe