Research on immunological gene expression

Fluidigm and the immune system

Research activities are focused on the innate immune system, the first line of defense against invading microorganisms. The expression of coding and non-coding RNA is characterised as response to infection or inflammation in livestock. 

The goal is to perform research that facilitate discovery of new biomarkers for diagnostic, prognostic and monitoring uses, and pinpoint targets for future development of therapeutics entities.

Present activities include characterisation of the innate host response in pigs after infection with viral and bacterial lung pathogens as well as development and optimization of methods for identification, characterization, and quantification of coding and non-coding RNA in tissue samples, cell cultures, and blood.

Within the past year, we have optimized our high-throughput real-time quantitative PCR platform, Fluidigm. We have designed, validated, and implemented hundreds of primer assays for measuring the expression of a wide range of immune factors in pigs, cattle, horses, sheep, chickens, and mice.

Gene activity in lung infections

Respiratory infections cause enormous economic losses to Danish and foreign pig producers. Recently, we have identified which genes are up and down regulated in lung, tonsil, liver and spleen after infection with Actinobacillus pleuropneumoniae; a Gram-negative bacterium causing an acute and very rapidly evolving pneumonia in pigs. We investigate the interaction between host and pathogen during the infection by measuring the expression of genes encoded by both organisms in the immediate surroundings of the invading pathogen, in order to get a more comprehensive understanding of the infection dynamics.

The transcriptional activity of immune factors including cytokines, pattern recognition receptors, and acute phase proteins as well as non-protein coding RNA (microRNA) is quantified during infection and inflammation. MicroRNAs are small single-stranded RNA molecules. They do not code for proteins, but they can regulate the activity of protein-coding genes. Expression of human miRNAs is well studied. However, expression of non-coding RNA and its importance for a well-performing immune response is considerably less studied in livestock such as pigs, horses, and cattle. We examine the role of non-coding RNA during influenza infection in the pig - especially in relation to its regulation of the pig innate immune system.

Fluidigm

In order to analyze expression levels of mRNA coding for immune factors and microRNA, we use several methods including Fluidigm qPCR. By using the Fluidigm platform, it is possible to measure the specific activity of each of 96 selected genes in a large number of samples simultaneously and thereby obtain a comprehensive picture of the immunological response relatively quickly and inexpensively. This high-throughput qPCR system is based on microfluidic and combines 96 samples with 96 primer sets for 9216 simultaneous qPCR reactions in a single run within the dynamic array chip (BioMark, Fluidigm). The qPCR is highly cost-effective as it requires only small volumes of reagents. Sample material is combined with primer, buffer, and enzyme in separate qPCR reaction chambers each holding  a volume of less than 10 nl.

The test material - cDNA from purified RNA - is added to the wells on one side of the chip (one sample in each well) and up to 96 selected primer pairs directed against specific genes (one primer pair in each well) is added to wells on the other side of the chip. In this way, we can analyze 96 samples for up and down regulation of 96 different genes in one qPCR run, which takes approximately 2½ hours.

Courses and student projects

At the course Assay Technology, 24101 it is possible to get an introduction  to the various steps involved in the purification and quality measurement of RNA, optimization of cDNA synthesis, qPCR reactions, and subsequent data analysis of gene expression data.

We are always looking for enthusiastic students, who want to work with gene expression analysis in infection and inflammation.

Senior Researcher

Kerstin Skovgaard
Senior Researcher
DTU Vet
+45 35 88 63 62