Dear Biostars.
I am trying to inform a kinetic model using 2 experiments (control and KD). The issue being experiment 1 is from microarrays and experiment 2 is from qPCR data. The input into the model must be compatible.
I have attempted to convert the qPCR data to something similar to the microarray data, however my methods are not straight forward, despite outputting values which make sense based on RNAseq data and visualising the qPCR data. The main issue is that different formula's need to be used if the genes are going up or down. I'll show my calculations for 2 genes, X (decreasing) and Y (increasing). I'll also include a link to a spreadsheet which will show the numbers and formulas clearly.
Experiment 1 (microarray) is under control conditions.
X Y
Day 0 6.2 13.2
Day 1 7.0 9.8
Day 2 9.1 9.8
Experiment 2 (qPCR) is under control and KD conditions.
X_KD X_control Y_KD Y_control
Day 0 4.3E-08 1.3E-06 6.6E-06 7.1E-06
Day 1 2.8E-05 2.7E-03 5.5E-06 3.3E-06
Day 2 4.4E-03 1.3E-02 2.5E-06 6.0E-07
First I do 1/n.
X_KD X_control Y_KD Y_control
Day 0 23097027 736497 150607 139897
Day 1 34867 3702 178746 294910
Day 2 2232 766 391773 1656099
Next I need to normalise Experiment 2 using the control. The initial values for the control and KD must be the same for kinetic modelling, so I assume Day 0 is equal to 1.
An ongoing issue I had was that the same formula was not working for X and Y, because X decreases and Y increases. The 1/n made this confusing. In the end, 2 different formula's had to be used if a gene was increasing or decreasing in the KD condition. I am aware that this sounds dodgy, hence this question.
For X (decreasing) I did KD/Control and Y (increasing) I did Control/KD.
X_KD Y_KD
Day 0 1 1
Day 1 9.4 1.6
Day 2 2.9 4.2
Finally, I used the microarray values to estimate the KD values.
For Day 0 it was 1 * Day 0 value, so the model will have the same start point for each gene.
Again I had to use slightly different formula's for X and Y for the reason above. If the gene was decreasing (X) I did microarray value / normalised qPCR value and if the gene was increasing I did microarray value * normalised expression value.
X_KD Y_KD
Day 0 6.2 13.2
Day 1 0.7 16.2
Day 2 3.1 41.8
Sorry for the very long question, but this issue has confused me for a while now. I believe the estimated values I get at the end are accurate because the RNAseq shows the genes change in this direction and when plotting the average qPCR numbers, the trends look similar to what numbers see here. However, the fact that 2 different formula's need to be used makes me concerned.
Any insight in how I can improve this, or another method I could use would be very appreciated.
Thanks, Krutik
