Mrna Transcript Quantification In Yeast With Single-Molecule Fret
BIOPHYSICAL JOURNAL(2017)
摘要
Quantitative determination of mRNA copy number distribution is essential for understanding phenotypic variability of single cells. Long mRNA transcripts (u003e 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.
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