RNA FISH is a powerfull tool to measure not only the amount of mature mRNAs in the cytoplasm (or other compartments) but also to asses the amount of nascent transcripts that are still at the transcription site. These nascent transcripts are RNAs that are still in the elongation or termination step of transcription (i.e. have not moved out ot the transcription site, where the gene is located).
Typically, in order to estimate the the number of nascent transcripts, one needs to calculate the average fluorescent intensity of individual RNAs (that are located outside the transcription site) and then divide the intensity of the transcription site by that average.
For example, if we take the average intensity of a single mRNA as 1, and the intensity at the transcription site equals 5, it means that we have 5 RNAs at the transcription site.
However, the desnse co-localization of these nascent transcripts prevent accurate estimate of the number of transcripts. Furthermore, the simple comparison of intensity looses information on 3D, or even 2D arrangement of the transcripts at the transcription site, and may also pose a problem with assesing the quality of the FISH signal.
Now, the groups of Zimmer, Darzacq & Bertrand developed a new tool that allows much more accurate measurments of nascent mRNAs at transcription sites. Their program, FISH-quant (can be freely downloded here), utilizes two methods to measure the number of transcripts: 1. integrated intensity of the transcription site and 2. reconstruction of the transcription site signal by iterative superposition of weighted point-spread function of the RNA spots.
The results in their report at Nature methods show that the simple method used before underestimates the number of RNAs at the transcription site. Their program should facilitate better measurments of transcription sites or other FISH-signal aggregates. For example, they mention P-bodies & stress granules in their paper, but there are other RNA granules such as neuronal granuls and germ cell granules.
Of course, this program should be similarly useful for live cell imaging of transcription sites or other RNA granuls.
I can think of other uses. For instance, the number of ribosomes on an mRNA (using super-resolution with fluorescently tagged ribosomes) or the nuber of RNAs in microvesicels and more.
I may even try it myself in a FISH experiment I’m doing now. If I use it, I’ll report on its ease of use.
Mueller, F., Senecal, A., Tantale, K., Marie-Nelly, H., Ly, N., Collin, O., Basyuk, E., Bertrand, E., Darzacq, X., & Zimmer, C. (2013). FISH-quant: automatic counting of transcripts in 3D FISH images Nature Methods, 10 (4), 277-278 DOI: 10.1038/nmeth.2406