Advanta RNA-Seq workflow helps advance research

What is the primary focus of your research, and why did you go in that direction?

As an intestinal stem cell biologist, I am interested in understanding the genetic mechanisms that control stem cell maintenance and differentiation. Your entire gut lining is renewed every week in a stem cell-driven process. We really want to understand the basic biology of intestinal stem cells and learn more about the genes that control their behavior, ultimately leading us to develop translational approaches to stem cell-based therapies for human disease and injury of the intestine. To this end, we have a number of projects focused on elucidating genetic processes that underlie stemness and developing microscale cell culture models to establish a clearer understanding of stem cell-driven regeneration pathways in homeostasis and injury.

While organoids that grow in 3D hydrogels have revolutionized in vitro studies of the intestinal epithelium, there are limitations that prohibit physiological relevance and efficient analyses. To address this, we developed 2D epithelial monolayer systems that serve as ideal experimental models, are scalable and can be amenable to high-throughput analyses. We then created a microtech device to examine the intestinal epithelial response to oxygen deprivation or other environmental signals over time.

Can you give us an example of how the experiments you design in the lab translate to potential therapies for human disease?

Even though we have identified several pathways that regulate the maintenance and proper differentiation of stem cells, one thing we don’t fully understand is how environmental factors, disease states or injury events impact this process from start to finish. One way we are studying this is to examine how stem cells function during ischemia, which is loss of blood flow that can lead to reduced oxygen, or hypoxia, in cells and possibly result in cell death. Ischemia can occur due to stroke in the gut, blood flow interruption during surgery, twisting of the gut or necrotizing enterocolitis. If we can understand how and when stem cells react to ischemic events, we could potentially propose therapeutic interventions to restimulate the stem cells and repair the gut.

Using the gut-on-a-chip microtech device, we developed a hypoxia-on-a-chip environment allowing accurate oxygen level adjustment and detailed measurement of the magnitude of oxygen concentration in a single system at the same time. This approach enabled us to observe stem cell expression patterns with varying magnitude and duration of hypoxia.

Why did you pick the Advanta™ RNA-Seq NGS Library Prep Kit for your project?

After initial determination that we could elicit a response from stem cells to a hypoxic event, we wanted to analyze stem cell response at different time points and elucidate how different gene expression pathways reacted to allow the cells to survive in a hypoxic environment, or not. For us, the Advanta RNA-seq workflow was vital to our studies because we were challenged with only being able to retrieve a small number of cells from our model for subsequent analysis. The culture chamber in the gut-on-a-chip system is very small–the entire five-well platform is smaller than a microscope slide–and we only get about 50,000 cells that generate a confluent monolayer. This translates to small amounts of cellular material and small amounts of harvested total RNA to use in the library prep protocol. Conventional RNA-seq systems simply cannot achieve what we need on this system. Also, in order to perform all technical and biological replicates necessary for these experiments, conventional systems become very expensive. The Advanta system was a game changer, enabling us to use these microdevices and characterize as many transcriptional responses as we can while managing costs.

How did the Advanta RNA-Seq NGS Library Prep Kit help in your hypoxia-on-a-chip research?

The Advanta system generated excellent data quality from our samples with low cell numbers, providing a really nice overview of what was happening to these stem cells during a hypoxic event. For example, we obtained >30 million reads per sample, 98.5% aligned to the human genome reference and 80.2% aligned to the transcriptome reference. Principal component analyses further demonstrated that samples treated under similar conditions clustered together. This level of clean data helped us immensely in pulling out differential gene expression patterns that we could use to generate hypotheses for how the cells were responding to hypoxia over time.

In fact, we were able to determine the top 100 most differentially regulated genes at each measured time point for both normoxia and hypoxia and compare the data across the three time points. Duration of hypoxia on the stem cells induced stereotypical early and late gene expression patterns, where the number and type of unique and differentially regulated genes change over time to increase the cells’ chance of survival.

What’s next for you regarding this project?

With what we were able to accomplish in these experiments, we are excited to move forward with additional studies. One of the original reasons we did this work was to see how long stem cells can survive. Performing additional functional experiments to investigate our hypotheses for how certain pathways protect cells from hypoxia will determine whether we can ultimately lengthen stem cell life during hypoxic events.

Learn more about the Advanta RNA-Seq NGS Library Prep Kit