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After applying the calculated correction factors, the qRT-PCR data were mostly consistent with the microarray results, displaying a significant correlation between the log ratios of the considered genes in the qRT-PCR and the corresponding log ratios in the microarray analysis with correlation coefficient of 0.86 and significance P-value <0.01.
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Additional real-time validation of the microarray results is displayed in Table S3.
Twenty differentially expressed genes were validated by qRT-PCR, and the qRT-PCR results displayed a similar tendency in variation as the microarray results.
To display an overview of the microarray results, we clustered the transcripts according to fold change using R language, which revealed that the expression level of most transcripts did not change in either part 1 or part 2 (Fig. 4D).
In order to validate the microarray results, 10 different loci that were either upregulated, downregulated or displayed no changed in expression profile in the microarray experiment were selected for the qPCR verification.
In the M. bovis-infected group, STAT1 showed significant reduced relative expression (-1.27-fold) based on real time qRT-PCR results but displayed significant increased relative expression (+1.28-fold) according to the microarray results.
Results of RT-qPCR analysis of the same panels displayed linearity over the same range of copy numbers as the microarray results.
Linear regression analyses displayed highly significant correlations (r = 0.982) between qRT-PCR and microarray results for the 12 evaluated genes (see Additional file 2), confirming the validity of the microarray results.
Two genes (IFNGR2 and TLR4) displayed significant expression differences between the two sample groups based on the microarray results, but were not significantly different based on real time qRT-PCR data analysis.
For relative quantification and microarray results validation, we selected primer sets and probes, matching sequences present in the IMAGE human cDNA clones of the nylon microarrays with those displayed on the web portal of Applied Biosystems.
This was in line with the positive microarray results.
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