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: RNA FISH, Fluorescence In Situ Hybridization, Saccharomyces cerevisiae, Budding yeast, Transcription, Single molecule
Background
Precise quantification of the transcript profile of single cells is possible by single molecule Fluorescence In Situ Hybridization (smFISH). This procedure gives good signal to noise by targeting a single mRNA molecule with multiple fluorescently labeled DNA oligo probes (Raj and Tyagi, 2010). Using this scheme, mRNA of length shorter than 200 nucleotides cannot be detected. However, in most experiments, the absolute transcript copy number is less informative than the relative copy number. To detect short transcripts or sequences, a short single DNA oligo probe can be used. The detection efficiency of a single probe is greater than 50 percent when using a single fluorophore to count mRNA (Wadsworth et al., 2017).
Materials and Reagents
Equipment
Software
Procedure
Data analysis
The Matlab Image Processing Toolbox was used to analyze the three-dimensional images. In cases where the researcher is unfamiliar with coding, we recommend FISH-quant for its rigor and user-friendly GUI. For systems with very non- uniform illumination, Corrected Intensity Distributions using Regularized Energy minimization (CIDRE) (Smith et al., 2015) can be used to flatten the images. Many functions in the Image Processing Toolbox can be accelerated by simply converting them to a gpuArray () in Matlab with a compatible graphics card (e.g., Nvidia Geforce 1080). An outline of the algorithm used to locate cells and spots is as follows:
Notes
Recipes
Q: The concentration at the end of reagent.
Acknowledgments
This protocol has been adapted from Raj et al. (2010). This work was supported by Georgia Institute of Technology startup funds, GAANN Molecular Biophysics and Biotechnology Fellowship, and the National Institutes of Health grant (R01-GM112882).
Competing interests
The authors declare no conflicts of interests or competing interests.
References
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