Figure 7.

Northern blot validation of the microarray results. (a) Comparison of total RNA and poly(A)⁺ RNA for northern analysis. 12 μg total RNA and 2 μg poly(A)⁺ mRNA from both the schizont (s) and trophozoite (t) stages of P. falciparum were blotted and probed with a PCR fragment overlapping the genomic sequence corresponding to oligonucleotide ID M11919_1, which represents the 41 kD antigen (p41), fructose-bisphosphate aldolase (PlasmoDB v4.0 annotated gene ID PF14_0425). (b) Total RNA northern blots were probed with PCR probes overlapping the corresponding oligonucleotide elements ('gene specific') for each gene. The selected gene set included a probable calcium-transporting ATPase (PlasmoDB MAL13P1.61), plasmodial heat-shock protein (PlasmoDB PFI0875w), a member of a family of conserved hypothetical proteins (PlasmoDB PFI1445w) and an ORF with sequence similarity to a sodium-and chloride-dependent taurine transporter (PlasmoDB MAL13P1.130), lactate dehydrogenase (PlasmoDB PF13_0141), and a heat-shock protein homolog of Hsp70-3 (PlasmoDB PF11_0351). The northern blot membranes were stripped and reprobed with the fructose bisphosphate aldolase control probe. Northern blot expression levels were measured by a phosphorimager. For each gene-specific probe, the measured ratio of trophozoite to schizont was divided by the ratio measured for aldolase on the same membrane. The average ratio from six replicate hybridizations on the microarray, including fluorophore reversal, is shown in the lower panel. The ratios express fold enrichment in either the trophozoite stage versus schizont stage (t) or schizont stage versus trophozoite stage (s).