Biological validation of the Oct4 RT-LATE-PCR assay: Oct4 expression in the two lineages of blastocysts. Real-time LATE-PCR plots generated by Oct4 templates in the following samples: expanded blastocyst after RT (blue line, RNA + DNA), expanded blastocyst without RT (pink line, DNA only) and single TE cell after RT (green line, RNA + DNA). Whole blastocysts contain Oct4 RNA (as shown by the delta between the pink and the blue line) due to the presence of the ICM. Oct4 expression is downregulated in the TE (the green line crosses the threshold very close to the 2-copy marker; two Oct4 template copies per cell are due to the presence of Oct4 genomic DNA). The positions of the white-to-red arrows correspond to known copy numbers in a genomic DNA standard, as color-coded; the placement of the green arrow was calculated based on this standard. Inset: Oct4 expression in the ICM isolated from an early blastocyst. Half of the ICM was used for the "+RT" determination (blue line) and half was used for the "No RT" determination (pink line). The probe used in all these experiments was an Oct4-specific molecular beacon conjugated to TET.