specific primer (globin: 0.5 iM). The PCR reactions were performed using a PTC-200

thermocycler (MJ Research, Watertown, MA). To ensure specific amplification, a hot

start PCR was employed by adding 1 IU Taq DNA polymerase (Invitrogen, Karlsruhe,

Germany)Gibco) at 720C. The PCR program employed an initial step of 97C for 2 min

and 720C for 2 min (hot start) followed by different cycle numbers (see Table 1) of 15 sec

each at 950C for DNA denaturation, 15 sec at different temperatures for annealing of

primers, and 15 sec at 720C for primer extension. The last cycle was followed by a 5-min

extension at 720C and cooling to 40C. As negative controls, tubes were prepared in which

RNA or reverse transcriptase was omitted during the RT reaction.

 The RT-PCR products were subjected to electrophoresis on a 2% (w/v) agarose

gel in lx TBE buffer (90 mM Tris, 90 mM borate, 2 mMEDTA, pH 8.3) containing 0.2

tg/ml ethidium bromide. The image of each gel was recorded using a charge-coupled

device camera (Quantix, Photometrics, Minchen, Germany) and the IP Lab Spectrum

program (Signal Analytics Corporation, Vienna, VA). The intensity of each band was

assessed by densitometry using an image analysis program (IP Lab Gel). The relative

amount of the mRNA of interest was calculated by dividing the intensity of the band for

each transcript by the intensity of the globin band for each embryo. To circumvent the

problem that the differences in the relative abundance of the transcripts were due to

different cell numbers of the blastocysts analyzed, the relative abundance of each

transcript for each embryo was divided by the mean total cell number for that treatment

and multiplied by 100. The value for mean total cell number for embryos in the replicates

used for RNA analysis were 131.8 cells (n=96) for control embryos and 117.7 cells (n =

76) for control embryos. For each pair of gene-specific primers, semilog plots of the