I've recently performed Affymetrix microarrays using the "MoGene 2.0 ST" Chips in order to analyze the differential expression in a mutant group compared to a control WT group ( n = 6 biological replicates for each group). I used the Expression Console and the Transcriptome Analysis Console (Affymetrix) for the analysis of the microarrays. Now I am validating these results by performing qPCRs with random genes, detected by the microarrays with various fold changes (FCs). I use a combination of housekeepers and the ddCt method for quantifying the qPCR results.
So far all of the genes I have analyzed via qPCR show the same direction of differential expression (up or down), however the FCs I get from the qPCR are always greater than the ones I observe in the microarrays, yielding up to 10-fold bigger FCs (for the strongest up- or downregulated genes) than the ones shown in the microarrays. Is this to be expected? Has anyone had a similar exprerience or a suggestion how should I interpret this?
Many thanks!
Hi Dilyana
In my experience it could be possible. Although the two methods are comparable, the used technologies are different and both might be biased by a lot of issues (efficiency of primers in RT-qPCR, different cDNA synthesis, and so on). In the past, I validated data from microarray and RNA-seq by using RT-qPCR and sometimes the fold-change were very different, but I considered the target approach (RT-qPCR) as the more precise method. I've got best results by using Rest2009 as software to calculate the fold-change. In addition, check carefully the p-value of both array and RT-qPCR. A nice reading on the topic: Morey, J. S., Ryan, J. C., & Van Dolah, F. M. (2006). Microarray validation: factors influencing correlation between oligonucleotide microarrays and real-time PCR. Biol Proced Online, 8(1), 175-193.
Hi Dilyana.
I've also found the same as you (I've used MoGene 1.0 ST chips and ddCt method) . I think that the most important is to obtain the same direction of expression. As microarray and qPCR are different techniques, with different approaches, it is very unlikely to get very similar results.
To be even more sure about the up/downregulation of your genes, you could select some for protein analysis by WB, if possible and feasible. But I would be satisfied with qPCR showing the same direction of expression.
Regards
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