# How to normalize with 2 housekeeping genes? - (Jan/24/2006 )

Hello,

tell me, what is the best way to normalize my target gene Ct values to the expression of to housekeeping genes?

Setup:

1 run for Target

1 run for Housekeeping 1

1 run for Housekeeping 2

Normally I would divide Target/Housekeeping gene to get the expression ratio. But what do I do with 2 housekeeping genes? Geometric mean first? But with what: the Ct values? Or do I calculate Target/House1 and Target/House2 and than the mean of the two results?

I could not find a paper (except for Vandesompele et al. 2002) that would describe an excact method how to do this?

Thanks in andvance, Tobias

Hi Tobias,

I am solving the same problem right now. Did you find any way how to proceed?

Thanks,

Paja

tell me, what is the best way to normalize my target gene Ct values to the expression of to housekeeping genes?

Setup:

1 run for Target

1 run for Housekeeping 1

1 run for Housekeeping 2

Normally I would divide Target/Housekeeping gene to get the expression ratio. But what do I do with 2 housekeeping genes? Geometric mean first? But with what: the Ct values? Or do I calculate Target/House1 and Target/House2 and than the mean of the two results?

I could not find a paper (except for Vandesompele et al. 2002) that would describe an excact method how to do this?

Thanks in andvance, Tobias

I did in so far as I came across the qbase excel add-in.

You can find it here: http://medgen.ugent.be/qbase/. It from the group of Vandesompele, that is very familiar with qPCR.

I don't really like this program, because the interface is too complicated imho, however they exactly explain their calculation methods in the provided manual.

So what they do is FIRST taking the geometric mean of the relative housekeeping gene expressions. AFTERWARDS they use this value to normalize target gene expression to HKG expression.

I adapted this procedure for my Excel sheet. So first I use the LightCycler program to import the external standard curves (normalization for efficieny) for target genes and HKGs and afterwards I take the geometric mean of the normalized HKGs and caclculate the ratio =Target gene / geo.mean (HKGs).

Ciao, Tobias

Great! That's exactely what I started doing I just wasn't sure if it was right or wrong...

Thanks a lot!

Paja

You might be interested in the new qBasePlus software, which is much more user friendly, and works 20x faster. qBasePlus is the professional successor of qBase and is being developed by Biogazelle.

You can find it here: http://medgen.ugent.be/qbase/. It from the group of Vandesompele, that is very familiar with qPCR.

I don't really like this program, because the interface is too complicated imho, however they exactly explain their calculation methods in the provided manual.

So what they do is FIRST taking the geometric mean of the relative housekeeping gene expressions. AFTERWARDS they use this value to normalize target gene expression to HKG expression.

I adapted this procedure for my Excel sheet. So first I use the LightCycler program to import the external standard curves (normalization for efficieny) for target genes and HKGs and afterwards I take the geometric mean of the normalized HKGs and caclculate the ratio =Target gene / geo.mean (HKGs).

Ciao, Tobias