Understanding Multiple Sclerosis Expression using Gene Knockouts

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Customer Spotlight: Dr. Alessandro Didonna, Assistant Professor, East Carolina University

Multiple sclerosis (MS) affects almost a million people in the United States. This disease of the central nervous system has unpredictable and variable symptoms, and its underlying cause remains unknown. Understanding the molecular basis of neuroinflammatory diseases, such as MS, is the focus of the lab of Dr. Alessandro Didonna, PhD, Assistant Professor in the department of Anatomy and Cell Biology at The Brody School of Medicine at East Carolina University. Didonna transitioned to his current role at ECU in 2021, having been in a faculty position at UC San Francisco prior. He earned his PhD at the International School for Advanced Studies in Italy, where he studied the effects of prion replication on neuronal signaling.

The Didonna Lab aims to connect the missing pieces between neuroinflammation and neurodegeneration in human disease. The bulk of their research effort is done on multiple sclerosis and its mouse model, experimental autoimmune encephalomyelitis (EAE). The group uses mouse strains with gene knockouts to determine what roles specific genes may play in development or progression of disease. The experimental mice are genotyped by PCR. They then use the Azure 300 Imaging System to image SYBR Safe-stained agarose gels to analyze the PCR products.

The Azure 300 is a chemiluminescent Western blot and gel imager with a 9.1 MP camera that easily images at the click of a button. Capture software is accessed through a built-in touchscreen that gives users the option to either utilize preset protocols with a comprehensive list of typical detections, or intuitively generate their own custom protocols. These custom protocols can combine any excitation source, emission filter, image overlay, lens aperture, exposure time, focus, distance to the camera, and resolution.

Female scientist next to Azure 300 Imager
Cindy Martines, undergraduate student in the Didonna Lab, posing next to an Azure 300 imager. She is a junior double majoring in Cellular-Molecular Biology and Neuroscience at ECU.

Using the Azure 300 to image Coomassie blue-stained gels and chemiluminescent Western blots

As part of the analysis of the effects of gene knockouts in EAE, the group analyzes gene and protein expression in the experimental mice. Students and research staff frequently use the Azure 300 to image Coomassie blue–stained protein gels and chemiluminescent Western blots.

“We are very pleased about its sensitivity to detect very low signals, as well as its user-friendly software interface,” says Dr. Didonna. “This is very useful when it comes to training students who have very limited lab experience.”

Using direct-mirror-free detection, Azure Imagers provide high-resolution imaging that’s perfect for publications. A CCD sensor is placed close to the sample to maximize light collection efficiency and sensitivity. Users are also easily able to change the sample to optics distance using an adjustable height shelf.

Intuitive capture software controlled by a wide touchscreen

The Azure 300 is an upgradeable chemiluminescent imager that provides accurate and fast chemiluminescent, UV, blue light, visible light, and trans white detection. It has the sensitivity, dynamic range, and linearity needed for quantitative blot analysis.

Studying changes in gene expression using qPCR

The lab carries out “omics” approaches to the study of disease states, including mass-spectrometry proteomics and RNA-seq gene expression profiling. A Cielo qPCR System from Azure Biosystems is used for qPCR experiments to study changes in the expression of candidate genes or to confirm gene expression changes observed by RNA-seq.

"We have been very impressed with the accuracy of the Cielo 6 in quantifying gene expression. This is especially important when you run technical replicates to obtain robust results."

Rather than a single, stationery light source and detector system, the Cielo utilizes 16-well simultaneous excitation and signal detection that ensures uniform data acquisition. The Cielo is able to demonstrate reproducible quantitation down to a single copy of the target sequence.

Male scientist next to Cielo qPCR system with open plate
PhD Student, Jonathan Carver, pictured next to the Cielo. Carver was the first author of a paper titled "Lipid metabolism is dysregulated in endocrine glands upon autoimmune demyelination," that published in May of 2024 in the Journal of Neuroimmunology using results from the Cielo.

Gene expression refers to functional gene products (usually proteins) resulting from a specific gene being transcribed or “expressed”.

This is a multi-step process where gene regulation can occur at any step along the path including transcription, post-transcription, translation and post-translation.

Learn more about gene expression >>

Recent publication from the Didonna Lab

The Didonna Lab recently published some of their work on MS in the Journal of Neuroimmunology1, titled Lipid metabolism is dysregulated in endocrine glands upon autoimmune demyelination. In this study, Carver et al carried out an analysis of the transcriptome of the adrenal gland and ovaries in EAE using RNA-seq. The Cielo was used to validate the RNA-seq results, as well as to study the dynamics of neuropeptide gene expression in the hypothalamus during disease progression.

The study found dysregulation of pathways involved in fatty acid and cholesterol biosynthesis and supports the idea that effects on the neuroendocrine system are an important aspect of this autoimmune disease. “We have been very impressed with the accuracy of the Cielo 6 in quantifying gene expression,” states Dr. Didonna. “This is especially important when you run technical replicates to obtain robust results.”

Ready to expand your research horizons? Check out the capabilities of the Azure 300 Imager and discover how it can enhance your results by clicking here.

Check out what the Didonna Lab is up to and stay updated on their research into immune responses and neurodegenerative processes in chronic human disease on their website.

The Cielo makes quantitative qPCR easy as A-B-C

Azure is confident you will be impressed with the ease of use and performance of the Cielo has to offer. We'll arrange to send your lab a system to use for one week, without any obligation, absolutely FREE.
Azure Cielo qPCR system

SOURCES

  1. Carver JJ, Pugh BA, Lau KM, Didonna A. Lipid metabolism is dysregulated in endocrine glands upon autoimmune demyelination. J Neuroimmunol. 2024;391:578366.

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