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In addition to microarray validation, the qPCR methodology was used to test several presumptions.
For microarray validation, the ΔCt values were calculated for each gene in each sample, log2 transformed and plotted against its corresponding least-square means data from the microarray.
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The results of the MS-HRM microarray validation corroborated the microarray results for 21 of the selected target sequences (unpublished data [ 7]).
Further, independent tissue microarray validation for the first time revealed aggregation of aSyn in a subset of PDAC specimens, similar to its presentation in PD.
It is important to note that our RT-PCR microarray validation analyzed only the expression of the two key cytokines IL8 and MCP1 and did not include genes induced specifically by WT-CF compared to ΔPrtT CF. Based on the results of the microarray analysis, we hypothesized that A. fumigatus germinating conidia or CF activate MAPK signaling in A549 cells.
Microarray validation did prove the usefulness of the tool for further transcript quantification in Atlantic halibut.
Two of the MS-HRM assays used in the microarray validation experiments did not confirm the microarray results.
All DMRs positively validated in the microarray validation experiments were subjected to initial clinical validation screening.
We have screened 275 samples of breast cancer with the same MS-HRM assays used in the microarray validation process as well as 74 DNA samples from breast reduction surgeries (representing healthy controls).
The microarray validation by independent quantitative real-time PCR (qRT-PCR) on some of the genes was performed (data not shown) as described in our earlier publications [ 68, 70].
For microarray validation, 22 strains from the American Type Culture Collection or other culture collections and 50 milk samples were tested.
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