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Genetic Profiling for Blood Brain Barrier Diseases – Life in Real-time with Dr. Joel Pachter

2,572 views · Published 20 September 2012 · 4:51 · Indexed 22 October 2026

Channel: Thermo Fisher Scientific · 2012 · Science & Technology

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To learn more, visit: http://www.lifetechnologies.com/arcturus Dr. Joel Pachter, Professor of Cell Biology at the University of Connecticut Health Center discusses how he was able genetically profile micro-vessels to monitor changes during the progression of inflammatory disease such as Multiple Sclerosis. Take a look at how Dr. Pachter used the Life Technologies laser capture microdissection (LCM)/qPCR workflow to get a better understanding of how to begin to develop pharmacatherapies to address diseases associated with the blood brain barrier.

I'm Joel Pachter, I'm a professor of cell biology at the University of Connecticut Health Center in Farmington, Connecticut. My laboratory mainly focuses on the blood/brain barrier and in CNS inflammatory diseases, like multiple sclerosis.

We're actually using laser capture and qPCR  to help genetically profile micro-vessels to see what changes occur during the progression of inflammatory disease. We realized that the vasculature was very heterogeneous and in order to better model that system, we had to get a better understanding of how the, how diverse the micro-vessels were in situ- in the brain and in the spinal cord.  And that's what ultimately drove us to use laser capture for that purpose.

The first challenge was how to get those vessels out of a tissue section- and how to identify the vessels.  So we had to develop immuno-staining protocols that would clearly identify the vessel populations that we wanted, and also help us identify the cells that we didn't want to get, and with laser capture we could do both. We wanted to stain for an androgen that was present throughout the vasculature, and then use size as a means of selecting the specific types of vessels that we wanted.  We also wanted to avoid contaminating neural tissue, and so we supplemented the immunohistochemical staining protocol with an immunoflourescence protocol, which allowed us to view in live time two androgens that were labeled, and being able to extract one and leave the other behind.

The IR laser capture process is very unique to the Arcturus System.  The UV systems, which just use the UV system alone have a problem in that the UV light can, uh, introduce energy into the system that results in cross-linking of RNAs to proteins which makes it difficult to extract.  

We've evaluated other platforms that are limited by the downstream applications that we can couple them to, whereas the Arcturus System is not. We knew that the qPCR was a good platform to go by because even the traditional hybridization based arrays have to be confirmed by qPCR in the end  A focused array seemed much more efficient to focus on the panels of genes that we thought were involved in the processes that we were looking at.  So for the, uh, mouse immune array panel, it seemed perfectly suited for our type of analysis.  

The benefit was time.  Before we were simply doing 2 to 3 genes at one time, which ultimately took years to be able to elaborate a profile.  With the TLDA analysis, we're able to analyze 384 genes at one time, and so, within about 6 hours from beginning to end starting the LCM process to finishing the TLDA analysis, we could get 384 genes.  And so the efficiency was right up front.

My experience with Life Technologies has been very positive because it was as easy platform to couple the laser capture with their downstream applications being all housed really within one company.  So I was able to speak with field operators in all aspects of the process.  And that made the development of our immuno-LCM TLDA analysis all that much better.  

We're making considerable headway in understanding how the vasculature of the brain, is really integrated.  And how certain vessels respond to certain stimuli and are involved in certain disease processes and also even normal processes- this will help us better generate and develop new pharmacatherapies to specifically address the problems that we've known about with regard to the blood brain barrier.

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