MRNA Transcript Quantification in Yeast with Single-Molecule FRET

Abstract

Quantitative determination of mRNA copy number distribution is essential for understanding phenotypic variability of single cells. Long mRNA transcripts (> 500 nt) can be detected using single-molecule Fluorescence In Situ Hybridization (FISH), where the target mRNA is hybridized with a large number of fluorescently labeled DNA probes. One downside to this method is the inability to interrogate short targets. We demonstrate a novel mRNA detection protocol based on Fluorescence Resonance Energy Transfer (FRET) which uses only a single pair of singly labeled DNA probes, each 24-nt long. Compared to conventional FISH, our method features higher signal-to-noise and lower cellular auto-fluorescence, thus effectively eliminating false positives. Using this technique in yeast, we estimate that ∼50% of transcripts can be detected. Our method will provide a reliable and efficient means to quantify relative levels of short mRNA transcripts in single cells.