Single Molecule Imaging of RNA In Situ
This protocol describes a method to image individual mRNA molecules in situ. About 50 oligonucleotides complementary to different regions of a target mRNA species are used simultaneously. Each probe is labeled with a single fluorescent moiety. When these probes bind to their target, each mRNA molecule becomes so intensely fluorescent that it can be seen as a fine fluorescent spot. Several different mRNA species can be detected in multiplex imaging using differently colored probe sets for each species. An automated image-processing program is used to count the number of mRNA molecules of each species that are expressed in each cell, thus yielding single-cell gene expression profiles.
Key wordsmRNA visualization FISH Single molecule detection In situ hybridization MATLAB image processing Gene expression profiling
This work was supported by a grant from the National Institutes of Mental Health (MH079197). Authors acknowledge help from Salvatore A.E. Marras in probe labeling and purification.
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