Validation of repeat RNA-seq data by quantitative RT-PCR. (a) qRT-PCR expression of IAP sequences in WT and Lsh−/− cell showing major transcriptional de-repression at IAP-gag and IAPEz-int. (b) Similar to (a), marked de-repression of mouse major satellite transcripts. (c) Short interspersed element sequences show low expression in all cell types (note scale). (d) LINE-1 5’UTR and ORF1 transcripts show little de-repression in mutant cell types. (e) GAPDH expression loading control for cell types analysed showing appropriate levels of this constitutive transcript. -rt shows undetectable signal in the absence of reverse transcriptase. (f) Indicated repeat expression levels in Dnmt3b+/− and Dnmt3b−/− MEFs. Blue = WT fibroblasts; green = Lsh−/− fibroblasts; orange = Dnmt3b+/− MEFs; brown = Dnmt3b−/− MEFs. Experiments represent triplicate analysis. See Figure S6 in Additional file 1 for qRT-PCR primer locations. Expression level units are arbitrary and are all normalised to GAPDH expression levels. A.E.U, arbitrary expression units, SINE, short interspersed nuclear element; UTR, untranslated region.
Dunican et al. Genome Biology 2013 14:R146 doi:10.1186/gb-2013-14-12-r146