Simultaneous amplification of Oct4 and Xist genomic DNA even at low template copy numbers. A FAM-conjugated molecular beacon was used to monitor Xist amplification (red lines; fluorescence scale on the left of each graph) and a TET-conjugated molecular beacon was designed for Oct4 template quantification (blue lines; fluorescence scale on the right of each graph) in a duplex LATE-PCR assay. (A) Serial dilutions of male mouse DNA, containing respectively (left-to-right) 1000 genomes, 100 genomes and 10 genomes, show consistent co-amplification of Oct4 and Xist DNA at all three dilutions tested. The CT values of standard scales such as this were used for template copy number quantification in unknown samples. Fluorescent signals arising earlier (smaller CT values) indicated the presence of more templates than later-arising signals. (B) Oct4/Xist LATE-PCR analysis of a single blastomere isolated from a 4-cell stage embryo. The real-time fluorescent signals reveal the presence of both Oct4 DNA (known to be present in two copies per cell) and Xist DNA (one copy in male cells, two copies in female cells). RT was not performed in this experiment, thus the sex of this embryo could not be determined by Xist RNA quantification.