An in situ hybridization approach distinguishes M-opsin and L-opsin mRNA.

(A) Alignment of human M-opsin and L-opsin mRNA. Green bar = nucleotide difference. Horizontal pink and blue lines = location of in situ hybridization probes. (B) Alignment of portions of exon 5 from M- and L-opsin. In situ hybridization probes target mRNA sequences, indicated by blue (M-opsin) and pink (L-opsin) boxes. Green arrowheads indicate 8 nucleotide differences. Dots indicate nucleotide alignment between the opsins. (C–H) HEK293 cells probed for M-opsin mRNA (blue) and L-opsin mRNA (pink). Insets = schematic of transfected plasmid. Cells that did not express M-opsin mRNA or L-opsin mRNA were not quantified. (C) Quantification of transfected HEK293 cells expressing M-opsin mRNA only, L-opsin mRNA only, or M-opsin mRNA and L-opsin mRNA for the conditions in (D–H). (D–H) Brightfield images of cells with: (D) No plasmid transfected. (E) Transfection of a plasmid driving M-opsin. (F) Transfection of a plasmid driving L-opsin. (G) Transfection of either a plasmid driving M-opsin or a plasmid driving L-opsin independently and then the cells were mixed. (H) Transfection of both a plasmid driving M-opsin and a plasmid driving L-opsin. (I) Visualization of M-opsin mRNA, L-opsin mRNA, and M-/L-opsin protein (black) in HEK293 cells transfected with both a plasmid driving M-opsin and a plasmid driving L-opsin. M-opsin (blue) and L-opsin (pink). Blue arrow indicates a cell expressing M-opsin mRNA only. Pink arrows indicate cells expressing L-opsin mRNA only. Purple arrow indicates a cell expressing both M-opsin mRNA and L-opsin mRNA. Black arrow indicates an untransfected cell. Cells were identified based on nuclear Hoechst staining (S1A Fig). With this combined RNA in situ hybridization/immunohistochemistry approach, the mRNA signal was reduced, when compared to the mRNA signal observed when RNA in situ hybridization was conducted alone (Fig 1H). (J) Quantification of M-/L-opsin mRNA and M/L-opsin protein expression in transfected HEK293 cells (I). Original data sets are in S1 Data. (K) Quantification of M-/L-opsin mRNA and M/L-opsin protein expression in adult human retina (L). Original data sets are in S1 Data. (L) Visualization of M-opsin mRNA, L-opsin mRNA, and M-/L-opsin protein in cone cells in an adult human retina. M-opsin (blue) and L-opsin (pink). No cones co-expressed M-opsin mRNA and L-opsin mRNA. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. Cell boundaries were determined by identifying layers from a nuclear Hoechst stain (S1B Fig) and analyzing opsin protein immunohistochemistry signal from the ONL to the OPL.