qPCR on low to non-existant template - (Dec/11/2007 )

Hi all,

I'm going to try to show that a silenced gene is not being expressed, or is very lowly expressed in my cells. I don't understand how to show this using real time PCR - if there is no expression, it won't amplify. How do I show that there is really no expression as opposed to the reaction failing? Perhaps real time PCR isn't such a great method to show this??

Can someone shed some light on how to do this?!
Cheers,
Dave

IMHO you can show this nicely by qPCR. I would simply incorporate some kind of internal control in your PCR reactions eg. you spike a cloned gene on a plasmid in every reaction and do a multiplex assay for your target and the gene on the plasmid. Or you do a multiplex reaction with your HKG in one tube. So you can rule out failed reactions (=reactions with no amplification at all).

But for this, you'll need differentially labeled fluorescent probes.

I agree. qPCR is definitely the most appropriate way to show knock-down

Thanks for the comments, I can see how it would work with multiplexing and fluorescent probes.

I was thinking along the lines of using Sybr green and looking for a reduction in CT values. Is it still possible with this method?

I think so, although the data isn't as 'hard' so it would require more repetition for you to be really sure, with extra controls as well.