Gene expression control in neuronsCustom LNA™ Detection Probes
Dr. Antonella Riccio
Dr. Catia Andreassi
Antonella Riccio’s lab at the MRC Laboratory for Molecular and Cellular Biology, UCL, in London studies neuronal gene expression and local protein synthesis in axons. Her group is trying to understand the triggers that induce translation of axonal mRNAs. With these questions in mind, Catia Andreassi, Senior Research Associate, has been working on mRNA localization in axons of primary sympathetic neurons using ISH, together with other approaches. Results of these studies have recently been published in Nature Neuroscience
1. What is the current research going on in your lab?
The work in the Riccio lab focuses on control of gene expression in neurons. Specifically two main areas of research are pursued: the epigenetic modifications that influence gene transcription in developing neurons, and the targeting and local translation of mRNAs in axons of neurotrophin-dependent neurons.
2. Why did you choose to use LNA™ mRNA detection probes from Exiqon?
The project I was working on, whose results are published in Nature Neuroscience (PubMed
), focused on the characterization of the role played by the neurothrophin NGF in the transport and local protein synthesis of the IMPA1 mRNA in the axons of primary sympathetic neurons. It was therefore instrumental to the success of the project the visualization of mRNA in subcellular compartments. I tried several FISH protocols, including the use of biotin-labeled oligonucleotides and riboprobes for the detection of the IMPA1 mRNA in axons, but given the extremely low amount of mRNA present in axons, the sensitivity and specificity of the other methods were not sufficient to obtain a nice signal above background level. This problem was completely circumvented by the use of LNA™ probes.
3. What label and detection method did you choose?
I used custom made biotin-labeled LNA™ probes. The FISH signal was amplified using the tyramide signal amplification system (Perkin Elmer).
4. How has it worked so far?
Brilliantly for all the LNA™ oligos I tried (designed to detect GFP, IMPA1 and a novel isoform of IMPA1 called IMPA1-L, mRNAs).
5. What were some specific challenges in your project?
Regarding this specific FISH experiment: First, the cell culture system: primary cultures of sympathetic neurons grown on glass (as needed for optimal observation at fluorescent microscopes) detach very easily during the FISH procedure. Second, the substrate on which the cells grow (collagen) gives high background, so performing hybridization and wash steps at high temperatures (possible with LNA™ probes but not with other DNA oligos) is mandatory to get a clean signal. Third, again because of the high background and low amount of mRNA, the optimization of the amplification step is very important.
6. How did you overcome them?
1) I carefully optimized the amount of collagen used to coat the glass coverslip. 2) I performed hybridization at 50ºC and washes at 55ºC in high stringency buffer. 3) I tried some chromophore and found that Atto565-conjugated (Sigma) or FITC-conjugated tyramide gave the best signal.
7. How do you feel about the in situ hybridization results you obtained?
Personally, I think they are comparable to the best ones obtained by other researchers so far.
8. Which new questions follow up experiments did follow the results you obtained?
Having identified (and confirmed by several methods, including FISH with LNA™ probes) a new isoform of IMPA1 mRNA in axons, I am now characterizing RNA-binding proteins responsible for the transport and localization of this mRNA isoform in axons of primary sympathetic neurons. I also plan to perform more FISH experiments to determine the presence of other novel mRNA isoforms in axons.
9. What advice would you give to researchers who want to get started in mRNA detection?
I would suggest trying several protocols and to be very meticulous in control experiments.
10. What would you tell a colleague about why they should work with Exiqon?
I enjoyed very much my collaboration with Exiqon. Their technical support is very efficient, the turn around time for the production of the LNA™ oligos quite good, and I believe they strive to give the best possible service to their customers.