4 replies to this topic

[TV2013]

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

[Mimoza]

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

[pcrman]

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.

[TV2013]

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

[TV2013]

Pcrman,

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