Investigating Dysregulated X Chromosome Inactivation in Human B Cells and Plasmacytoid Dendritic Cells by RNA FISH

RNA fluorescent in situ hybridization (RNA FISH) has been widely used to monitor the allelic expression of primary transcripts and/or non-coding RNAs in individual cell nuclei. In particular, this method is a gold standard to establish which X-linked genes undergo or escape X chromosome inactivation in female mammalian cells by combining probes specific of the X-linked transcripts of interest with probe specific of the long-non-coding XIST RNA that coats the inactive X (Xi) territory. Here we describe the dual RNA FISH protocol we have developed to track (1) the coating of the Xi by the XIST RNA and (2) the expression of the X-linked TLR7 gene on the active and inactive X chromosomes, in human plasmacytoid dendritic cells (pDCs) and in female B lymphocytes induced to differentiate into plasmablasts or pre-Antibody Secreting Cells (pre-ASC) upon activation of either TLR7 or TLR9.