SYBR Green Ct values of 18S, beta actin and GAPDH

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health science
health science's picture
SYBR Green Ct values of 18S, beta actin and GAPDH

Dear All,
We get Ct values of house keeping genes around 20 cycles. Ideally 18S should have come around 9 cycles and GAPDH at 13 cycles. Can anybody guide me in the same. We design primers from IDT site.
cheers

Ckis
Ckis's picture
There is no place that a Ct

There is no place that a Ct "should" come out. Where the threshold comes out is dependent on the starting material you put into the reaction. The more starting material, the more you will amplify and the sooner your threshold will be reached. A lower Ct simply means you have a lot of starting material. Nothing wrong with a Ct of 20 for either of those.

By the way, a Ct of 9 is too low. The sensitivity of the assay works best between Ct 12-35. Below 12 and you have too much starting material which could interfere in amplification. Later than 35 and you have too little. Curves coming off at Ct 36-38 or later are probably due to non-specific events.

health science
health science's picture
Dear Ckis,

Dear Ckis,
Thanks a ton for ur commets