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A previous study has indicated that the VAS is a valid ratio measure [ 27].
After filtering low quality reads and removing sequences with N, 17,142,420 (valid ratio 88.43 %) clean reads from A1, 16,728,306 (valid ratio 86.04 %) from A3 and 18,712,824 (85.10 %) from A9 were obtained, respectively.
Only genes exhibiting substantial changes of RNA levels upon acid treatment (at least threefold signal intensities above local background, expression ratios ≥2 for at least at one time point, more than three valid ratio values) were considered, and the final data were analyzed using GeneSight (Biodiscovery).
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To be stringent we considered in this test only genes with more than 4 valid reconstructed ratios for the indirect dataset and only genes with all valid ratios for the direct dataset.
This would explain the lower fraction of valid ratios in the direct comparisons, where a number of transcripts having an expression level near detection cutoff in one tissue would eliminate those genes from the analysis when labeled with Cy3.
Only protein-coding genes with a fully annotated translation start and end, and a valid dN/dS ratio are included for consideration (see Methodology section " Gene length and dN/dS ratio ").
If the multiplicative model and the framework are valid, the ratio between the sample mean and the variance of (log(N)) should remain constant over time.
Genes without a valid dN/dS ratio were removed (i.e. without any observed synonymous polymorphisms according to dbSNPv138 and EVS).
For all three disease-associated-gene lists, we mapped the gene symbols to their official HGNC gene symbol (and discarded the ones that could not be mapped), retained only protein-coding genes with a fully annotated translation start and end, and a valid dN/dS ratio.
Precision of reconstructed ratios was also evaluated in terms of the average variance of all valid expression ratios measured across the range of expression intensities.
The cut-off points of TDC derived from scores from the 0 4 points scales were also valid for ratios in subsequent scores of the intensity of the predominant pain in the masticatory system from a 100 mm Visual Analogue Scale (VAS; part of anamnesis, see Appendix).
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