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Hi everyone,
I am having trouble with my nested PCRs. I used siRNA to knockdown a gene and their recommended method of checking for down-regulated expression is nested PCR.
I have done the first round of PCR. After normalisation to the house keeping gene (GAPDH), the transcript levels of my supposedly "down regulated gene" didn't show any difference between the control and siRNA treated sample. I assume this is because I didn't get a single product.
I didn't get a single product. Must I purify this to obtain amplicons at the right size (~1000bp) before I begin my second PCR to get the best result?
I don't understand why nested PCRs are used for monitor gene expression knockdown....would be really great if someone could give me some explanation/direction!
Thanks very much!
Hedgehog
I am having trouble with my nested PCRs. I used siRNA to knockdown a gene and their recommended method of checking for down-regulated expression is nested PCR.
I have done the first round of PCR. After normalisation to the house keeping gene (GAPDH), the transcript levels of my supposedly "down regulated gene" didn't show any difference between the control and siRNA treated sample. I assume this is because I didn't get a single product.
I didn't get a single product. Must I purify this to obtain amplicons at the right size (~1000bp) before I begin my second PCR to get the best result?
I don't understand why nested PCRs are used for monitor gene expression knockdown....would be really great if someone could give me some explanation/direction!
Thanks very much!
Hedgehog