New RNA Extraction Approaches Overcome Agarose Barriers in Lung Slices

Precision-cut lung slices allow researchers to study living human lung tissue outside the body, preserving the different cell types and structures that make up the organ. But a material that helps scientists create these slices can also make it difficult to study the genetic activity within them.

Researchers at Yale School of Medicine (YSM) have developed two approaches to solve that problem, increasing the amount and integrity of RNA that can be recovered from precision-cut lung slices, or PCLS. In a recent study published in the American Journal of Respiratory Cell and Molecular Biology, the researchers show how modifying the RNA extraction process can overcome interference from residual agarose, a gel-like substance needed to prepare the lung slices.

To create PCLS, researchers inflate donated human lungs with agarose to provide enough structural support to cut the otherwise soft, porous tissues into thin slices. The resulting samples retain multiple types of lung cells and structures, allowing researchers to examine how cells interact and respond to experimental treatments.

The fact that there are different cell types within the tissue is really useful for studying how cells interact and respond to one another because the cells within our organs are built to communicate with each other. That's something PCLS allow us to study."

Reina Rangel, first author, research associate, YSM Section of Pulmonary, Critical Care and Sleep Medicine

But agarose presents researchers with a trade-off.

"The agarose is extremely helpful in that it enables us to cut the PCLS in the first place," Rangel says. "But it becomes a barrier that prevents us from doing certain downstream things."

In particular, residual agarose can interfere with extracting RNA, the molecules that researchers analyze to understand gene expression. Conventional extraction produced RNA quantities below the study's detection threshold and substantially lower RNA integrity than either of the alternative approaches the researchers tested.

Rangel approached the problem by considering agarose's biological origins. Because agarose is a polysaccharide derived from seaweed, she wondered whether tools designed to extract RNA from plant tissues, which also contain complex carbohydrates, could work for lung samples.

"I thought about it more like a biologist," Rangel says. "I was interested in what agarose actually is and whether there was anything about it that I could exploit. Agarose comes from seaweed, so I thought maybe using a plant kit to extract the RNA could help."

While Rangel pursued the plant-based approach, associate research scientist Joan Marti-Munoz, PhD, the last and corresponding author of the study, looked for a way to remove the agarose before extracting the RNA. Marti-Munoz implemented an agarose-dissolving buffer commonly used to purify nucleic acids in gel electrophoresis. Both methods improved RNA recovery, with the agarose dissolution method producing the highest RNA yield and integrity.

"I looked for a way to dissolve the agarose in PCLS before extracting RNA and found that a buffer routinely used in gel electrophoresis worked well," Marti-Munoz says.

For the team, one of the most important improvements was reliability. Previously, researchers could set aside several lung slices for RNA analysis, only to lose some because the extraction failed.

"Now, when we're designing our experiments, we can say, 'we want five samples to go out for RNA extraction,' and we'll actually get five RNA samples," Rangel says.

More reliable, intact RNA allows researchers to use PCLS for sophisticated analyses, such as RNA sequencing, while comparing how tissue from the same human donor responds to different experimental treatments. That could be particularly useful for studying pulmonary fibrosis and evaluating potential therapies.

"Obtaining high-quality RNA is essential for meaningful gene expression studies, and our method helps researchers achieve this goal," Marti-Munoz says.

By examining changes in gene expression, researchers can better understand how lung tissue responds to experimental drugs. For diseases such as pulmonary fibrosis, which causes scarring of the lungs, these analyses could help scientists assess whether potential treatments are having their intended effects.

"Human tissue is valuable for our research because when we're designing drugs, we want them to work in people," Rangel says. "Precision-cut lung slices enable you to have multiple samples from the same person, giving researchers better tools to answer what we think are very important questions."

Other Yale authors of the study include Sabina Anderson, Giuseppe Deluliis, Edward Manning, Farida Ahangari, and Naftali Kaminski.

Pulmonary, Critical Care and Sleep Medicine is one of 10 sections in the Yale Department of Internal Medicine. To learn more about Yale-PCCSM, visit PCCSM's website or follow them on Facebook and Twitter.