Figure 1
Multiplex Xist/Sry template detection in individual blastocysts processed with the single-tube PurAmp method. Sry (upper panel) and Xist (lower panel) real-time PCR plots generated by six single embryos at the blastocyst stage processed via PurAmp. All steps, starting with cell lysis, were performed by progressive dilution in the same optical-grade tube. Xist and Sry amplicons (genomic DNA plus cDNA when RNA was present) were detected simultaneously by using sequence-specific molecular beacons. Each color identifies a single embryo and is used in both panels. Female embryos (lines in red hues) were easily distinguished from male embryos (lines in blue hues) based on the presence of Xist RNA, which causes the female Xist signals to be much earlier than the male Xist signals, and on the absence of the Y-chromosome-specific Sry gene. Quantification of Xist and Sry copy numbers (given in the text) was obtained based on the CT of the real-time PCR plots (see Methods), using a genomic DNA standard curve as detailed in Fig. 2. The horizontal line in each chart indicates the threshold used to determine the CT values. The dashed vertical lines facilitate the comparison of Sry and Xist CT values in male embryos.