Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes.
Measurements of gene expression within single cells have revealed startling variability otherwise hidden in bulk measurements. Here, we present an in situ hybridization method capable of detecting individual mRNA molecules, thus permitting the accurate quantification and localization of mRNA within fixed sample. Our in situ protocol involves probing the target mRNA using a series of singly labeled oligonucleotide probes. This method is simple to implement and is applicable to a variety of biological samples. We also discuss some aspects of image processing required for analyzing the resulting data.
Raj
Arjun
A
Department of Bioengineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Tyagi
Sanjay
S
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D017403:In Situ Hybridization / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D015003:Yeasts / Q000235:genetics
D015345:Oligonucleotide Probes; D012313:RNA