standard curve - normalization - (Mar/21/2007 )

hi everybody
I'm new in this forum and i hope someone can help me! i'm doing ChIP for histone modifications and then i'm analyzing the sample by qRT-PCR ( Sybr green). My (big) problem is how to normalize my sample....someone told me to use the delta-deltaCt method, someone else told me to use the standard curve......now i do the standard curve for each IPed sample with its input and i normalize this value for the housekeeping gene....does it sound correct?
laura

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May I know what's ChIP?
But anyway, generally for normalizations you have to use housekeeping genes, and depending on a lot of other factors, you can choose to use any of the 3 most often used relative quantitation methods: delta delta CT, standard curve and Pfaffl method. The delta delta CT assumes that your amplification efficiencies for both target gene and housekeeping gene must be similar so that only the Cts are compared.
The standard curve and Pfaffl method are quite similar in that you need to run standard curves, but for the Pfaffl method you don't need to include the standard curves for each run you do. So using the Pfaffl method, you do a standard curve in triplicate of a normal, untreated sample with the housekeeping and target gene respectively. Take the efficiency. Then run your untreated and treated for each primer set. Take the Cts. Using his mathematical formula you can then get the fold change of your target gene in treated sample and untreated sample.

Chris

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