Single-probe RNA FISH in Yeast

Abstract

Quantitative profiling of mRNA expression is an important part of understanding the state of a cell. The technique of RNA Fluorescence In Situ Hybridization (FISH) involves targeting an RNA transcript with a set of 40 complementary fluorescently labeled DNA oligonucleotide probes. However, there are many circumstances such as transcripts shorter than 200 nt, splicing variations, or alternate initiation sites that create transcripts that would be indistinguishable to a set of multiple probes. To this end we adapted the standard FISH protocol to allow the use of a single probe with a single fluorophore to quantify the amount of transcripts inside budding yeast cells. In addition to allowing the quantification of short transcripts or short features of transcripts, this technique reduces the cost of performing FISH.

Keywords: Fluorescence In Situ Hybridization; Budding yeast; RNA FISH; Saccharomyces cerevisiae; Single molecule; Transcription.

Figure 1.. Comparing Epi and HILO illumination.

A. An epi-fluorescence microscope has the illumination incident (red line) on the sample through the objective and the entire volume of the sample is illuminated. This leads to poor signal to noise for single fluorophores since the widefield microscope collects out of focus light. B. An inclined illumination geometry has the illumination (red line) displaced radially in the back focal plane of the objective so that the light becomes a thin laminated sheet in the sample volume ( Tokunaga et al., 2008 ). This enables z-sectioning and increases signal to noise significantly.

Figure 2.. Schematic of the illumination path.

One or more lasers are coupled into a fiber optic cable using a fiberport. This fiber is fitted onto an X-Y translation mount and collimated by a short lens (focal length chosen by the field of view). The tube lens focuses the illumination on the back focal plane. When the X-Y position is adjusted away from the center of the back focal plane the beam becomes inclined through the sample volume.

Figure 3.. Contrast between strains.

A. One z-slice of a negative control for yEvenus mRNA (e.g., wildtype) is shown. All intensity is due to auto-fluorescence. The yEvenus probes are 26nt in length and labeled internally (sequence in Wadsworth, et al., 2017). B. One z-slice of a strain expressing a low copy number (< 20) of yEvenus mRNA transcripts per cell is shown. mRNA transcripts are targeted with a single Cy5 labeled DNA oligo probe.