A number of gene expression studies done in single cell organisms have shown that variable gene expression due to stochastic molecular processes can lead to phenotypic variability, and because of that genes must be regulated in an appropriate way in each cell. This becomes much more intricate during C. elegans development when multiple cells also have to communicate with one another via various molecular signals susceptible to intrinsic and/or extrinsic fluctuations but still have to divide timely and adopt their proper cell fates.
In the need of quantitative measurement of endogenous gene expression to tackle these problems, our lab has recently developed an in situ hybridization method which allows us to detect individual copies of mRNA molecules in C. elegans embryos and larvae as diffraction-limited fluorescent spots (Raj et al., 2008 ). Using the available nucleic acid sequence of the target mRNA, we designed 48 different singly labeled ~20bp long oligonucleotide probes. Having a large number of probes targeting one transcript ensures the signal-to-noise ratio to be much less sensitive to non-specific binding of the probes and only the target mRNA molecules appear as a bright fluorescent spots in the image.
Our new method has the following advantages: 1) we can measure discrete quantity and location of mRNA molecules in fixed animals with preserved shape, 2) it is quick – custom designed oligonucleotide probes can be commercially synthesized in less than a week, 3) multiple genes can be probed simultaneously – so far we were able to reliably detect up to three different genes by labeling each probe with three different fluorophores in addition to DAPI nuclear staining, and 4) we can measure endogenous mRNA level and avoid artifacts of genetic constructs which often lack unidentified key regulatory regions such as UTRs, intronic sequences, etc.
More details of our in situ hybridization technique including detailed protocols and probe designer algorithm are available online at http://www.singlemoleculefish.com/
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