RNA-FISH for Xist and Tsix Detection

The samples for RNA-FISH were prepared as previously described [4 (link)]. In brief, for Xist detection, the pXist12.9 plasmid containing the majority of the Xist cDNA was used (kindly gifted by T. Sado). For Tsix detection, the region (around 7 kb) of the Tsix locus from chr X: 103,448,873 to 103,455,853 was amplified by PCR and the products were subjected to nick translation (Abbott Laboratories). The region from chr X: 103,459,241 to 103,460,958 was amplified by PCR and cloned in the PUC118 vector (Takara), resulting in PCU118-Tsix1.7. The plasmid was also subjected to nick translation along with the PCR products of the 7 kb region. The FISH images were observed using a LSM510 laser scanning confocal microscope using C-.Apochromat 40x/1.2 W (Carl Zeiss).