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Ct value very high. What can I do to improve this ct? - (Feb/07/2013 )

Hi everybody

I work with Sybr to analyze 5-HT (Serotonin receptor) in rat hypothalamus. However, during the primer optimization the ct value for this gene has been very high (30- 32). We have already redesigned twice the primers for 5-HT and we got same result. Last experiment, we concentrated the cDNA pool samples for 1:5 and the ct didn't change.
I don't know what to do in this case.
Can anybody help me?
Thanks a lot


Don't you think it could be simply because your gene is very low expressed, if not at all?


How much RNA did you use for RT? You should consider increasing the starting RNA a bit. Using gene specific primers for RT reaction is another option.


I have already thought on this but I would like to be sure that is only this. Do you understand?



I have used 1ug of RNA for RT and my primer is specific for the gene.
Thank you