Tertiary screening in the procyclic form. Independent RNAi cell lines generated in PF T. brucei for dynein heavy chain (Tb927.3.930), panel A; TOR1 (Tb10.6k15.2060), panel B; TOR-like 2 kinase (Tb927.1.1930), panel C and a hypothetical ORF (Tb927.5.3260), panel D, were analysed for cell cycle progression defects following RNAi induction. Upper left of each panel: cumulative growth curves for each clone cultured in SDM79 with appropriate selective drugs [13, 14] and the absence (-tet) or presence (+tet) of 1 μgml-1 tetracycline. Lower left of each panel: real time PCR analysis of mRNA levels for each gene (using oligonucleotides detailed in Additional file 5 and standardised against a GPI8 control) at 96 hrs (dynein heavy chain and hypothetical ORF), 161 hrs (TOR-like 2 kinase) or 212 hrs (TOR1) post-induction. Middle of each panel: analysis of nuclei and kinetoplast numbers as determined over time by DAPI staining. Upper right of panels A and D and right of panels B and C: flow cytometry profiles of uninduced (-tet) and induced (+tet) cells at the time points indicated. The DNA content of each peak is given. Lower right panels A and D: examples of abnormal cells generated. Left, DIC image; right, DAPI image. Black bar: 5 μm. The number of nuclei (N) and kinetoplasts (K) in each cell is given.