Gene expression and molecular biology research in vision and ophthalmology presents unique challenges to researchers and clinicians due to the complexity of the eye. The eye consists of many structures containing specific cell populations, each exhibiting different expression profiles. Such an intricate organ requires a series of advanced methods in your molecular toolkit to unlock the mechanisms of degenerative eye disease.
Diagram of a Human Eye
Stellaris® RNA FISH can shed light on the location and amount of RNA in retinal cells
A recent publication from Sui Wang et al. applied Stellaris RNA FISH (fluorescence in situ hybridization) probes to measure mRNA levels within individual cells of retinal explants containing heterogeneous cell populations1. Using multiple oligonucleotides targeting a single RNA, Stellaris RNA FISH assays can quantify and localize RNA within tissue sections or cells while preserving morphology. Each fluorescently labeled probe collectively binds along the same target transcript to produce a punctate signal for an individual RNA molecule.
The authors sought to unravel the complex interactions required for the specification of different central nervous system cell types by examining whether the RORβ or Otx2 transcription factors were required for the activation of the Blimp1 gene1. Blimp1 is a transcription factor involved in determining the cell fate of rod versus bipolar cells in the retina. RORβ or Otx2 were targeted and degraded via shRNA knockdown. The shRNAs were placed 3’ to the EGFP gene, and Blimp1 mRNA from EGFP positive cells were quantified.
Fewer retinal cells had a high number of Blimp1 signals when RORβ expression was targeted. Likewise, compared with controls, significantly fewer retinal cells contained a high number of Blimp1 signals when the Otx2-shRNA was introduced. The authors concluded that RORβ and Otx2 were positive regulators of Blimp1 expression1. Because the levels of transcription factors can be important for developmental events, the quantitative nature of Stellaris RNA FISH makes it a valuable method for evaluating transcript levels in heterogeneous cell populations.
- A Gene Regulatory Network Controls the Binary Fate Decision of Rodand Bipolar Cells in the Vertebrate Retina. Sui Wang, Cem Sengel, Mark M. Emerson, and Constance L. Cepko. Developmental Cell. 2014. doi: 10.1016/j.devcel.2014.07.018