|Title||Visualization and analysis of mRNA molecules using fluorescence in situ hybridization in Saccharomyces cerevisiae.|
|Publication Type||Journal Article|
|Year of Publication||2013|
|Authors||R McIsaac, S, Silverman, SJ, Parsons, L, Xu, P, Briehof, R, McClean, MN, Botstein, D|
|Journal||J Vis Exp|
|Keywords||Algorithms, In Situ Hybridization, Fluorescence, RNA, Fungal, RNA, Messenger, Saccharomyces cerevisiae|
The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.
|Alternate Journal||J Vis Exp|