- All RCPs should be localized to the cytoplasm, however RCPs
can occasionally dissociate out of cells and generate RCPs on
the glass slide (such signals should be disregarded). - RCPs can be easily distinguished from cellular autofluorescence
by their defined brightness, shape and size. - Finally, RCP signal should be visible only in a single, fixed
fluorescence channels (checking other channels is a good mea-
sure to evaluate signal specificity). Figure 3 shows ACTB
mRNA genotyping in human BJhTERT and mouse MEF
fibroblast cells. Mutation within the gene allowed designing
two PLPs, which arms differ by only a single, 3^0 terminal base
(G/A in human/mouse). When PLPs for both alleles were
used together in cocultured cells, SNP specific signals were
exclusively generated in either human or mouse cells. - We applied the protocol to genotype and differentiatePCDH
XandYchromosome homologs in human embryo medulla
oblongata section. This study provided detailed insight into the
spatial distribution of PCDH11X/Y and NLGN4X/Y in
human developing nervous tissue. Observed patterns sug-
gested the significant development complexity in the male
central nervous system even if single cell expression data was
Fig. 3In situ SNP detection ofACTBmRNA in human BjhTERT and mouse MEF cell line. Detection ofACTBSNP
in human and mouse cell lines is represented as color-coded RCPs.Green: Detection of humanACTB;Red:
Detection of mouseACTBSNP. Size of the scale bar in theinset 50 μm
SNP Detection with Padlock Probes 221