Protocol Online logo
Top : Forum Archives: : Real-Time PCR

SYBR green - (Aug/18/2008 )

Dear all,

I am new to qRT PCR and am still reading about it. I will be using qRT PCR to measure the expression levels of a three member-gene families.

Could anyone help me with this question:
When we use SYBR green in real time, can the transcript levels of internal standard and target gene be distinguished? How?

Cheers, smile.gif



Unfortunately not. When I have used Sybr green and needed to monitor an internal control (ex. GAPDH) in addition to my target gene (ex. E2F1) I had to do two separate rxns in the same plate. Half for one and half for the other depending on how many samples I have. If you want to do them together you must multiplex using Taqman primers. When you multiplex however, you must also make sure the primers do not interfere with the amplification of one another. I had this to happen to me and it was not fun. Hope it helps!!!