Adding new reference gene ?

[GregoryMaes]

Hi all,

we study natural fish populations with various degrees of pollution and intend to analyse gene expression profiles at the individual level. Based on literature, we initially use done reference gene, but by the end of the study, this gene seemed to strongly correlate with pollution levels (data gathered later on). We have now found better ref genes for our analysis (after testing 7 new ones) but are struggling with a good way to link our new analyses to our initial ones.

We have a few plates with each time the old/bad ref gene (actin) and 2 GOIs per plate + Interplate calibrator. Now we ran two new plates with the two new ref genes for all samples + the IPC. How do we link those new plates ? All inds are run on one plate per ref gene, so I assume no IP calibration is needed?

We can't just make new columns with new Ref genes, as they were run on different plates and only the first ones should be calibrated with our IPC. Should this be done manually by pre-processing the plates first (leaving columns open ? ) and then exporting this step to new file for further preprocessing ? I assume when analysing 15 GOIs and 3 REF genes, people have to do this step (as you cant' put all genes in one plate) ?

So summarizing : 4 plates with each time old REF (actin) + GOI per ind + CAL (actin+ GOI), 2 plates with new REF genes + CAL (actin + new REF genes). How to link this ?

thanks

Greg

[Mikael Kubista]

Dear Greg,
GenEx has very popular methods to join data sets based on interplate calibrators, and I believe they should be suitable for your case.
Start by merging the two data sets containing 1) samples (S1) characterized for all GOI’s and the poor RG run together with Inter Plate Calibrator 1 (IPC1), and the same samples (S2) characterized for the new RG run together with IPC2. The merge data set will have a lot of missing data looking in short notation like (the CT values are there only for illustration and ND=Not determined):
GOI RG(old) RG(new)
S1 23 25 ND
S2 ND ND 26
IPC1 25 25.2 ND
IPC2 ND 26.2 27.2

Next normalize the data with Interplate calibrators in GenEx Editor. Using the IPC information GenEx normalizes the data to the same levels (i.e., as if they were measured in a common plate)
GOI RG(old) RG(new)
S1 23.5 25.5 ND
S2 ND ND 25.5

Since samples S1 and S2 were the same, the data can now be rearranged into compact form suitable for further processing and analysis:
GOI RG(old) RG(new)
S1 23.5 25.5 25.5

The column RG(old) can now be removed

GOI RG(new)
S1 23.5 25.5


Good luck