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Michael Thomsen, Mayuri Viswanathan, Samer Hattar; Single molecule fluorescent in situ hybridization in the mouse retina. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1765. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
High-throughput transcriptomic data sets often yield hundreds of interesting candidate genes for further study, but confirmation of this data in vivo often relies on methods of in situ hybridization that are time-consuming and technically challenging. To facilitate rapid, robust screening of gene targets from transcriptomic studies, we have adapted a protocol for single molecule fluorescent in situ hybridization (smFISH) for use in whole-mount mouse retinas. smFISH requires less than half the time of conventional in situ hybridization protocols and permits single transcript resolution and quantitation.
We used computational methods to design a library of 20-30mer oligonucleotide probes that tile >100 mRNA sequences of interest with minimal off-target binding. Each probe was flanked by universal primer sequences and gene-specific orthogonal priming sequences that permit amplification of probe sets for a single gene from the library. The total library of >12,000 oligos was chemically synthesized and suspended in a small volume of TE buffer. Probe sequences for each gene were amplified from the library using several rounds of PCR with gene-specific primers. Following amplification of probes for each gene, antisense fluorescent oligonucleotide probes were generated by PCR with fluorophore-coupled primers. Freshly dissected mouse retinas were quartered and lightly fixed in 4% PFA then permeabilized in 0.5% TritonX-100/PBS. Following permeabilization retinas were incubated overnight with fluorescent probes in hybridization buffer. After hybridization, retinas were washed several times in 10% formamide/2XSSC, mounted, and imaged immediately.
smFISH robustly labeled transcripts in the ganglion cell layer (GCL) of mouse retinas with little background fluorescence. Individual transcripts for more than 20 genes were visualized and quantified.
We have optimized a method of smFISH for rapid and reliable detection of individual mRNA transcripts in the mouse retina. This method has significant advantages over conventional in situ hybridization including shortened protocol time, single transcript resolution, and improved reliability.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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