Identification of microRNAs involved in the innate immune response microRNA NGS Service
Louise Brogaard works in the lab of Professor Peter M.H. Heegaard in the Section for Immunology and Vaccinology at the National Veterinary Institute, Technical University of Denmark.
The lab focuses on the innate immune system, the first line of defense against invading microorganisms. They are mainly interested in the expression of coding and non-coding RNAs in response to infection or inflammation using the domestic pig (Sus scrofa) as model system for human disease.
What is the main focus of the research conducted in your lab?
We work to uncover how the immune system responds to and copes with inflammation of various etiologies. A major aim is the characterization and validation of the pig, which is a major animal model in biomedical research, and the development of therapeutics for humans and animals.
How did you come to be interested in microRNAs?
In recent years, microRNAs have been found to play important regulatory roles in several pathologies, including the regulation of the human immune response to influenza A virus infection and control of viral replication. We are therefore interested in determining the corresponding processes in pigs in an ongoing effort to further establish this animal as a model of human diseases. In-depth understanding of the role played by microRNAs in the establishment and progression of influenza A virus infection will lead to the discovery of new biomarkers for diagnostics and disease monitoring. With the ongoing concerns regarding the possibility of an influenza pandemic caused by the emergence of new strains, this research is highly relevant and will have many practical applications.
What was the specific aim of your project?
The specific aim of the microRNA NGS project was to measure the expression of microRNAs in the lungs of pigs – both unvaccinated and vaccinated – at certain time points after they had been inoculated with an H1N2 influenza A virus strain. MicroRNAs from lung tissue from uninfected control pigs were also sequenced to serve as a baseline, so that the expression levels of microRNAs in infected animals could be compared.
What, if any, was your previous experience with NGS?
Prior to our collaboration with Exiqon Services, we had had a small subset of samples sequenced with another provider. This constituted a pilot study which aimed to determine if small RNA sequencing was a suitable approach for the sample material we had available.
What was the reason for choosing to perform the profiling using NGS?
We have excellent and unique material available from an in vivo influenza A virus infection study in pigs. This allows us to examine the microRNA response in the porcine host during the acute phase of infection as well as several weeks after infection. We found that NGS could provide the most comprehensive investigation of the global microRNA expression and regulation in these animals. Furthermore, NGS could give insight into microRNA expression profiles at controlled time points after infection – something which human studies have not been able to provide. The result from our study is a comprehensive profile of the temporal changes in the porcine lung of hundreds of microRNAs in response to influenza A virus infection.
How do you feel about your results from the microRNA NGS profiling experiments?
The quality of the data was very good. The Exiqon bioinformaticians had done an excellent job in mapping the sequence as well as post processing this data, which included tasks such as predicting novel microRNAs based on mapping data and mapping of viral reads, providing very useful results. The results from this NGS microRNA expression study will form the basis for my research in the coming years. Initial analyses demonstrate that the results represent a strong foundation for further examination of different aspects of microRNA involvement in influenza A virus infection – such as microRNA target prediction and GO analysis.
What were important aspects for you when choosing an RNA service partner?
It was important to find a service partner who could provide a clearly defined product – while still offering customization of the product – within a short time frame.
Why did you choose Exiqon as your service partner?
I chose Exiqon after talking with colleagues who have had positive experiences collaborating with Exiqon on microRNA expression profiling using miRCURY LNA™ microRNA Arrays and miRCURY LNA™ Universal RT microRNA PCR System.
What did you find to be the main benefits of the Exiqon NGS service?
Exiqon fulfilled the above mentioned criteria. I knew in advance which analyses we could expect Exiqon to perform, as well as the format in which the results would be delivered. They offered a great deal of flexibility; rather than having our sequencing data mapped to one of the default genomes included in Exiqon’s microRNA sequencing pipeline, it was possible to have the mapping performed to both the porcine genome as well as the genome of the influenza A virus strain used for inoculations.
Why would you recommend Exiqon and/or Exiqon’s NGS Service to colleagues?
Exiqon is very easy and professional to work with. I was continuously kept informed of the project progress and Exiqon provided excellent follow-up support upon data delivery. Importantly, the customer is not required to have the experience and computer power to analyze NGS raw data; Exiqon delivers mapped and quantified results in a report that is comprehensible for anyone.
What are the next steps in the current project and how do you plan to perform them?
In the coming months, qPCR will be used to validate the results obtained from the microRNA sequencing. This will enable us to investigate a large fraction of the microRNAs identified in our sequencing data as well as novel microRNAs not yet annotated in the porcine genome.