# Delta Delta Ct method for multiple reference genes? - (Jan/23/2011 )

Hi there

I was wondering if anyone could tell me whether you can use the delta delta Ct method for qPCR data analysis with multiple reference genes and if anyone would know how to go about this?

Thanks!

You can use it but consider that delta-delta is only an aproximation method, since it doesn't take into account different efficiencies of different genes. More housekeepings are used to make the results more accurate, but at the same time you just throw away your accuracy with a delta-delta.

You can use this Pfaffl equation:

Where E is the efficiency of each gene. If you put "2" as E, it's the same as delta-delta. But if you do a dilution series for each gene and actualy calculate the efficiencies, you may get more accurate results.

Now how to normalise to multiple references - instead of single E^deltaCP for one gene you make a geometric mean of all of them. That means if you have two housekeepings, in the denominator you will multiply each E^deltaCP and then take square root of the result. (if three, than again, multiply and then take cube-root, and so on).

The final equation is from this paper Hellemans, Mortier, De Paepe, Speleman, Vandesompele (2007) (but I had to ask mathematicians to explain in, because it looks horrible).