Examples of qRT-PCR validation of microarray expression data and DUX4 expression by RT-PCR. A. and B. By qRT-PCR on 20 - 40 ng of cDNA template, the relative expression of representative genes in FSHD vs. control myoblast or myotube samples from different individuals was determined for representative genes that displayed significant FSHD-linked differences on the microarray (Table 1). qRT-PCR signals for the test amplicons were normalized to those of M6PR. The average fold-changes deduced from the qRT-PCR and the microarray analysis are shown, and the p-values for the FSHD/control differences are indicated. C. and D. To detect DUX4-fl RNA (fl, full-length isoform, associated with FSHD), 400 ng of cDNA was used for real-time PCR as previously reported ; 20 - 40 ng of cDNA was used for the other amplicons. Results from different individual FSHD and control cell strains are shown. The samples used for DUX4 RT-PCR were the same as those used for qRT-PCR validation of microarray results.