Single molecule mRNA detection with FISH

Fluorescence in situ hybridization using a large number of singly labeled oligonucleotide probes lights up individual mRNAs

We have devised a variant of fluorescence in situ hybridization (FISH) capable of detecting individual molecules of the target mRNA. The procedure involves preparing a large number (at least 25, preferably more) of individual 20mer oligonucleotides, each singly labeled with a fluorophore, that each bind to a different region of the target mRNA. The binding of so many fluorophores to an individual mRNA renders it sufficiently fluorescent that one can detect the molecule as a diffraction limited spot using conventional fluorescence microscopy.

Applicability

We have shown that this method is applicable to a variety of biological specimens, including E. coli (Ido Golding, personal communication), yeast, mammalian cells, C. elegans embryos and L1-L2 larvae, Drosophila melanogaster wing imaginal discs, and primary rat hippocampal neurons. The primary concern when considering whether or not a particular sample is suitable for this method is the sample thickness: we have had difficulty getting the method to work with samples that are thicker than 15 microns, presumably because of the preponderance of out of focus light. It is possible that one may be able to obviate this restriction through the use of confocal imaging, but in our testing, the lower sensitivity of confocal imaging significantly hinders its applicability in this context.

Limitations

Other limitations arise from considerations of target mRNA length and GC content. As for length, we have detected mRNAs as short as 550 nucleotides in length (using around 20+ probes), but this is probably a lower limit and we recommend the use of at least 40 probes if possible, especially since experiments we have performed indicate that the number of probes required is sequence dependent. We have also found that our method works most optimally when the mRNA GC content is 40-50% overall, although we have often managed to obtain good signals when targeting mRNAs with higher GC content by using more stringent washing conditions.

Publications and contact information

Publications

Description of method

• Raj A., van den Bogaard P., Rifkin S., van Oudenaarden A., Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nature Methods, 2008 5(10) pp. 877-9.

Other papers using single molecule FISH

1. Femino A., Fay F., Fogarty K., Singer R. Visualization of single RNA transcripts in situ. Science, 1998 280(5363) pp. 585-90. [A very nice paper indeed!]
2. Vargas D., Raj A., Marras S., Kramer F., Tyagi S. Mechanism of mRNA transport in the nucleus. PNAS, 2005 102(47) pp. 17008-13.
3. Raj A., Peskin C., Tranchina D., Vargas D., Tyagi S. Stochastic mRNA synthesis in mammalian cells. PLoS Biology, 2006 4(10) pp. e309.
4. Maamar H., Raj A., Dubnau D. Noise in gene expression determines cell fate in B. subtilis. Science, 2007 317(5837) pp. 526-9.

Contact information

• Sanjay Tyagi, tyagisa(at)umdnj.edu
  PHRI/University of Medicine and Dentistry of New Jersey
• Arjun Raj, arjunraj(at)mit.edu
  Massachusetts Institute of Technology