Selection of the reference genes was based on the microarray results using an algorithm described in Popovici et al. (2009).
External corroboration of microarray results using PCR-based methodology also requires validation of appropriate internal reference genes for normalization of expression values.
This limitation has prompted genomics researchers to validate a statistically significant subset of their microarray results using a second technique, typically quantitative reverse transcription polymerase chain reaction (qRT-PCR).
The analysis of microarray results using a traditional 2-fold change in gene expression cutoff and a 10% false-discovery rate revealed a well-developed APR in catfish, with particularly high upregulation (>50-fold) of genes involved in iron homeostasis (i.e. intelectin, hemopexin, haptoglobin, ferritin, and transferrin).
We next validated the microarray results using qRT-PCR (Figure 2C).
We analyzed the microarray results using the statistical analysis of microarray (SAM) program, which yielded 45 positive hits.
Finally, we validated some of the microarray results using TaqMan®-based qRT-PCR on laser-microdissected neurons according to our previous study [12].
The five genes (ISG15, MX1, OAS1, IFI27 and IFI44) with the largest difference in fold change between NR and SVR groups were chosen to confirm the microarray results using real-time qPCR.
We had previously shown that Neurog3 activated Math6 expression in mPAC pancreatic duct cells [34], and we confirmed our previous microarray results using conventional RT-PCR and real time PCR (Figure 3A).
The 4 human miRNAs (miR-199a, miR-199a*, miR-200a, and miR-200b) with the largest difference in fold change between the F1 and F3 groups were chosen to validate the microarray results using stem-loop based real-time qPCR.
