This article has been reviewed according to Science X's editorial process and policies. Editors have highlighted the following attributes while ensuring the content's credibility: 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 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," says first author Reina Rangel, a research associate in the YSM Section of Pulmonary, Critical Care and Sleep Medicine. "That's something PCLS allow us to study." 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, Ph.D., 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.
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