We've tried to increase the concentration of cDNA and making sure that the correct ROX concentration was added. But we keep getting the same results, Beta Actin that is reading at 24 and above. Could you give me idea's as to how to lower or correct the readings for Beta Actin?
Have you checked you RNA? It could degraded or contaminated with inhibitors. Check your 260/280 and 260/280 readings, and run your samples on bioanalyzer to check for degradation. Also perform an inhibition curve using decreasing amounts or RNA, if you don't get a straight line (Ct vs concentration) than you have a inhibitor. Besides that, how are your primers? Do they show high efficiency? Do you have any primer dimers?
We did check the RNA and based on the 260/280 readings the RNA is not contaminated or degraded. As far the primers go they functioning correctly.
260/280 readings don't show you degradation. You need to run an TBE agarose gel and check 28S/18S ratio or run a Bioanalyzer analysis. The fact that primers are amplifying is not a indication of high efficiency. Did you calculate primer efficiency using a diluiton curve? I also emphasize the importance of a inhibition curve. If you increased cDNA concentration and your Ct didn't change that indicates that you have an inhibitor during your RT or qPCR step.
Thank you for you help. We'll try these and see if we can determine what exactly is going on.
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