Hi ALL, I am trying to do the qPCR and detect the BNDF gene in the mammary tissue. But the CT in my experiment is about 32. I know it means nothing! But in our previous experiment, the CT is about 27-29 cycles. I do not know what's wrong with my experiment. I am sure that I did exactly everything the kit I used said (iScript cDNA Synthesis Kit and iQ SYBR Green Supermix).
Please help me with this problem
Thank you so much
Decreasing the CTs can be achieved by loading more cDNA, using more efficient primer or in some cases by selecting a different Master-Mix. However, after making such changes you'll still need to confirm the linearity detection reactions against appropriate controls (e.g. a no-RT reaction, a no RNA reaction, and a no-RNA+no-RT reaction). Even a CT of 27-29 might not be quantitative. The limit-of detection needs to be determined empirically, and it's good to confirm you have specific amplification at such high CT's via melt-curve analysis and/or agarose gel electrophoresis.
Hi,
Delay in Ct could be due to suboptimal reaction conditions or due to degraded sample. Try a fresh sample extraction.
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