Detection of transcripts from both var2csa paralogs in P. falciparum parasites as seen by RNA-FISH. (a) Alignment of variable var2csa sequences serving as template for RNA probes (PFHG_05046.1 and PFHG05155.1) designed to discriminate between the two var2csa alleles in HB3CSA (asterisks indicate variable nucleotides between the two alleles in HB3CSA). The sequences of FCR3CSA and NF54CSA display high homology to PFHG_05155.1 in this particular region of the var2csa gene, with limited variability (denoted by arrows) allowing detection of the single copies in these parasites at the selected FISH stringency. Percent identical nucleotides between the sequences are displayed (with PFHG_05155.1 as 100%) to the bottom right. (b) Representative pictures of the hybridization patterns achieved with the two probes targeting var2csa mRNA in indicated parasites. The probe generated from PFHG_05155.1 is displayed in green; the probe towards PFHG_05046.1 in red and parasite nuclei stained with DAPI in blue. Probes towards antisense transcripts consistently showed no hybridization (data not shown). (c) Pictures representing the three scenarios observed for detection of var2csa allele transcripts in HB3CSA, with frequencies for each scenario (transcripts detected from both paralogs, transcripts detected from only allele 1, and transcripts detected from only allele 2) given as a percentage in each representative picture (n = 92). The great majority of simultaneously transcribed duplicated var2csa genes in single HB3CSA cells were exclusively accompanied by the observation of co-localization of the two transcripts in the nuclei. (d) Representative hybridization patterns achieved with the control probe targeting the kahrp gene (red) in nuclei (blue) of all the CSA-selected parasite lines used. For the var2csa hybridizations, the negative control probes towards antisense transcripts of kahrp revealed no detection of hybridization.