Quantprime

Significantly altered genes from premanifest HD versus controls were selected from the SAM analysis to confirm affymetrix findings by RT-qPCR. Validations were initially carried out using RT-qPCR on samples from cohort 1; both the samples with RIN ≥ 5 used for above affymetrix and on all remaining samples: 5 premanifest (3 male, 2 female); 6 stage II/III HD patients (6 male, 0 female); and 7 control subjects (5 male, 2 female). Further validations were then performed on a separate cohort (cohort 2): 9 premanifest (5 male, 4 female); 9 stage II/III HD patients (5 male, 4 female); and 10 control subjects (5 male, 5 female) (See Tables 123). For RT-qPCR experiments, all samples were run in triplicate for each target gene and housekeeping gene, and relevant negative and positive controls were run on each plate. Melt curves were inspected for all assays, with the Tm checked to be within known specifications for each assay. Sample assay data points were included in data analysis only if detected with Ct < 37 and at least 3 Ct values lower than the corresponding negative control [16] . Any data that did not pass these criteria were omitted from all further analyses. Primers utilised for RT-qPCR validations (see Table 4) were designed using either QuantPrime [17] , Primer3 [18, 19] or PrimerQuest from Integrated DNA Technologies (http://eu.idtdna.com/PrimerQuest).