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Fig. 9. Quantitative PCR analysis of Hox complex genes during embryoid body
differentiation. The E14 cells used for targeting (wild type), the
double-targeted Mll–/– cells and their FLPe rescued
derivatives were differentiated in suspension in the absence of LIF or
retinoic acid and harvested for RT-PCR analysis every second day. Each data
point is the average from three parallel experiments. That is, three culture
plates were differentiated, mRNA was harvested, cDNA was produced and Q-PCR
analysis took place for each data point in parallel. Results are plotted as Ct
values (one Ct value corresponds to a doubling of signal and all values less
than 31 were assumed to be zero, i.e. 31). Pbx1 served as a control for RNA
input. In our experience, the earlier activation of Hoxb2 and Hoxb5 seen in
the rescued cells compared with wild type represents an implicit degree of
variability involved in ES cell differentiation experiments, owing to either
clone-to-clone or slight onset of differentiation differences.
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