real time PCR with sybr green - having problems validating my knock down (Sep/14/2005 )

Any help would be greatly appreciated

I am currently using the biorad sybr green mix for my real time pcr to validate my knock down of a specific gene. However in 2 separate experiments, I see an INCREASE of my gene of interest rather then a knock down. This is observe in comparison of cDNA made from my knock down total RNA vs. wild type total RNA. I am using GAPDH as a reference gene and my GAPDH in the knock down and the wild type comes up within 1 cycle of each other, which I assume that it tells me the relative concentrations of my samples are about the same

both sets of experiments are done in triplecates.

anyone have any suggestions as what i am doing wrong?java script:emoticon(':(')

If there is only 1 cycle difference, the result is not significant.
Other than real-time PCR condition, I would check if the gene can really been knockdown.

Are you using transfection for siRNA delivery?
If yes, please check the transfection efficiency.
You can use the fluorescent siRNA oligos to check the transfection efficiency.

Dear RNAiNY,
Since you knock down seem to be increase which is a paradox......
I suspect that your knock down RNA in contaminated by you genomic DNA.
If your "RNA extract" also contain DNA, your relative quantification result is not valid.

Is your purity ratio A260:A280 < 2.0?
Did you carry out a PCR to your knock down RNA to show there is no amplification?
If your RNA is mix with DNA, you can treat your samples with DNase 1, but remember to deactivate it before you proceed to RT-PCR.

Best regards