With some computer savvy, and biochemistry knowledge, there are ways to detect genes activated in maturing photoreceptors in vivo, that are not detected by DNA micro-arrays or the average RNA-Seq method. While the general expectation was that photoreceptors need to be isolated, Dr Mitton used an alternative approach to leave rod-photoreceptors where they are, in the retina. Digesting retinas with proteases, and sorting cells over hours, will alter endogenous gene expression. So, we avoided this process entirely. We had done some ChIP-PCR analysis with the mouse retina and realized that ChIP (chromatin Immunoprecipitation) for RNA-Polymerase-II captures mostly rod-cell genomic DNA because more than 50% of the cells in the mouse retina are rods. ChIP is not a recovery efficient process and typically recovers only about 1 copy number of a DNA fragment location per 1-10,000 cells. Thus, when these retinal genomic DNA fragments are labeled and hybridized to tiling arrays, the fragments from rod photoreceptor nuclei are far in abundance over all other retinal cell types. Non-rod DNA fragments provide a low almost background signal on these arrays and differences in RNA-Polymerase-II binding between mouse retinas at post-natal age P2 and P25 reveals genes that are activated or shut down during the maturation of rod cells.
You can search for any gene of interest and view our maps of RNA-Polymerase-II binding regions, by clicking HERE: Photoreceptor Pol-II ChIP Map
This dataset was first described in our publication in Molecular Vision:
TEMPORAL CHIP-ON-CHIP OF RNA-POLYMERASE-II TO DETECT NOVEL GENE ACTIVATION EVENTS DURING PHOTORECEPTOR MATURATION. TUMMALA ET AL., 2010.
Dr. Mitton published this data in Molecular Vision to ensure free immediate access to the paper and the genome wide dataset for anyone, worldwide. Several other investigators have requested Pol-II data from our data-set to confirm new photoreceptor specific promoter regions in their gene of interest. Dr. Mitton provides high resolution tiling array data for their publications. Requests can be made to Dr. Mitton (firstname.lastname@example.org). For a good example of how to use gene specific data, see our own publication below, describing our discovery a previously unknown rod-specific alternative promoter for the Mef2c gene.