Reliable quantification of cell cycle-dependent mRNA abundance using fluorescence-activated cell sorting in Trypanosoma brucei.

Very little is known about cell cycle-dependent regulation of mRNA in Trypanosoma brucei, the causative agent of African sleeping sickness. Methods to synchronize cell cycle progression are inefficient or subject the parasites to non-physiological conditions and stress. We developed a fluorescence-activated cell sorting-based method to analyze steady-state mRNA levels in individual cell cycle phases. Normalization of the data was the most challenging problem because internal standards for cell cycle-regulated genes are not available for trypanosomes. Hence, we introduced an external standard (so-called "spike") to compensate for technically derived variations in processing cells and RNA samples. Validation of this method with a limited number of genes unraveled a transient up-regulation during S and G2/M phases for all mRNAs analyzed.