standard curve and differentiation asays - Real Time PCR (Aug/24/2006 )

I would appreciate responses on this one.

I am doing a differentiation assay on ES cells. I designed my universal cDNA from ES cells and differently staged embryos.

Validated my primers (15 gene set) and attempted(#1) to do a standard curve, currently using GAPDH as my reference gene. Did a 1/10 dilution of cDNAs and got nothing or very low CT values for my other genes (GAPDH was 20).

A post-doc from another lab (since I am introducing the technology to my lab) suggested to buy Universal RNA from Stratagene. I did that and last night.... had no amplifications for my genes except off course GAPDH. BTW

I don't know what to do. I want to use the relative method and is a standard curve really necessary?

In my case what should I use as my calibrator, ref cDNA mix for all or unique sample for each gene?

I need to get some results in 2 weeks!!

Thanks

Hi,

I think you should just need to stick with the relative method. The standard curve method is more for quantifying unknown samples against your samples in which concentrations are "known"

It sounds like your primers might be an issue. How did you design your primers?

Calibrators are like samples to which you compare against. For example, if you are studying the effects of drugs on cell types, the calibrator will be the sample in which no drug treatment was given.

Which real time machine are you using? I used the Applied Biosystems 7500 and the application specialists in my country are really helpful in troubleshooting and training me. If you are also using ABI, why don't you contact you local application specialist for help?

Hope these help!

kiwi

Hi Kiwi,

Thanks for your help.

I am uisng the Eppendorf MasterCycler, and the primers seem to be fine. I am doing an assay in which gene expression will go up then down or is down then will go up, throught the assay, therefore, I am not sure which is the best sample to use as my calibrator.

D