In situ detection of mRNAs using the PBCV-1 DNA ligase. (A) Reaction schematic: (i) The padlock probe and RCA primer hybridize to the fixed mRNA target. (ii) RNA splinted padlock probe is ligated by the PBCV-1 DNA ligase. (iii) The padlock probe is amplified within a primer-initiated RCA reaction, and detected with fluorescence probe complementary to a probe binding sequence (orange). (B) Representative image of HeLa cells with PP-1 (left) and PP-1n (right) after the padlock probe assay. Red, green, and blue are the counterstains for the RCA product, cytoskeleton, and nucleus, respectively. The scale bar represents 50 µM. (C) Box plot of the RCA dot count per cell obtained for the PP-1 and PP-1n padlock probe in 1000 HeLa cells. The whisker shows the cell-to-cell variation of the median RCA dot count per cell, whereas the outside bars show 2σ.
