I want to calculate a gene expression rate of 1 gene of interest and 2 reference genes (GAPDH, 18S) over different timepoints and accounted for primer efficiency. Since im working with RNA extracted from human whole blood, the reference genes vary among the timepoints, and Pfaffls equation does not work. But the quotient of the reference genes is consistent. Can I calculate the relative expression like this? (E = Primer Efficiency/100 +1)
Ratio to Reference gene: E^GOI / (E^GAPDH * E^18S)^0.5
(I adapted the reference gene calculation from Pfaffl)
And following the ratio compared to the control (timepoint 0)
Ratio to control: Ratio to reference gene timepoint 1/ ratio to reference gene timepoint 0
In comparison to Pfaffls equation I want to account first for the reference genes and second for the control.
Thank you for your answers!
It always seems easier to me to convert everything to relative quantities first, and THEN start normalizing. It makes the numbers more manageable, and you don't run the risk of losing track of what everything means (it's a lot easier, at a glance, to assess the difference between 0.25 and 1, than it is between 2^-8.19 and 2^-6.21, for example)
Get all your Cq values for a given gene (say 18S)
So RQsample = efficiency^(lowest Cq value - sample Cq)
For samples of
22.4
21.8
22.1
with an efficiency of 2, the lowest is 21.8, thus the RQs are
2^(21.8 - 22.4) = 0.66
2^(21.8 - 21.8) = 1
2^(21.8 - 22.1) = 0.81
Do the same for your second reference gene, and for your GOI, and then generate the geometric mean of your two reference genes to get your normalization factor (NF)
NFsample1 = sqrt(RQsample1Gene1 * RQsample1Gene2)
NFsample1 = sqrt(RQsample2Gene1 * RQsample2Gene2).
NFsample3 = sqrt(RQsample3Gene1 * RQsample3Gene2)..
Then simply divide your GOI RQ values by your NF values.
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