New RNA extraction techniques overcome barriers to help scientists better evaluate treatments for diseases such as pulmonary fibrosis.
RT’s Three Key Takeaways:
- Extraction Barrier Overcome: Researchers at Yale School of Medicine developed methods to overcome residual gel interference and significantly increase the recovery and integrity of ribonucleic acid (RNA) from living human lung tissue slices.
- Electrophoresis Buffer Advantage: While adapting a commercial plant-extraction kit improved yields, dissolving residual agarose with a standard gel electrophoresis buffer produced the highest RNA quantity, purity, and consistency.
- Therapeutic Screening Potential: Reliable genetic extraction enables healthcare researchers to evaluate how lung tissue from the same human donor responds to experimental therapies for respiratory conditions like pulmonary fibrosis.
Researchers at Yale School of Medicine have developed two laboratory approaches to improve the extraction of ribonucleic acid (RNA) from precision-cut lung slices, according to a study published in the American Journal of Respiratory Cell and Molecular Biology.
Precision-cut lung slices (PCLS) allow investigators to study living human lung tissue outside the body while preserving native architecture, cellular diversity, and intercellular signaling, according to the researchers. To produce the slices, scientists inflate donated human lungs with agarose, a gel-like substance derived from seaweed, which provides sufficient structural rigidity to slice soft, porous lung tissue into uniform sections.
“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,” said Reina Rangel, a research associate in the YSM Section of Pulmonary, Critical Care and Sleep Medicine, in a news release. “That’s something PCLS allow us to study.”
Overcoming Agarose Interference
Despite its utility during tissue preparation, residual agarose creates technical hurdles during subsequent molecular testing, according to the study.
“The agarose is extremely helpful in that it enables us to cut the PCLS in the first place,” said Rangel. “But it becomes a barrier that prevents us from doing certain downstream things.”
In conventional RNA extraction protocols, residual agarose interfered with molecular isolation, yielding RNA quantities that fell below detection thresholds and showed substantially lower integrity than alternative protocols, according to the study authors.
To resolve the issue, Rangel evaluated extraction kits developed for plant tissues, which contain complex polysaccharides similar to agarose.
“I thought about it more like a biologist,” said Rangel. “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.”
Concurrently, senior and corresponding author Joan Marti-Munoz, PhD, tested an agarose-dissolving buffer routinely used to purify nucleic acids during gel electrophoresis. Both strategies improved RNA recovery, with the dissolution buffer method yielding the highest overall RNA integrity and concentration.
“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,” said Marti-Munoz, an associate research scientist at YSM.
Implications for Pulmonary Fibrosis Research
The refined extraction protocols eliminate sample attrition, ensuring that tissue specimens set aside for testing reliably produce viable molecular samples, the researchers reported.
“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,” said Rangel.
According to the study team, high-integrity RNA permits advanced profiling methods, including RNA sequencing, to measure how tissue from a single human donor responds across parallel experimental drug exposures. This capacity is especially relevant for investigating progressive fibrotic lung conditions, such as pulmonary fibrosis, where cellular signaling and therapeutic response must be verified in intact human organ architecture.
“Obtaining high-quality RNA is essential for meaningful gene expression studies, and our method helps researchers achieve this goal,” said Marti-Munoz.
“Human tissue is valuable for our research because when we’re designing drugs, we want them to work in people,” said Rangel, in a news release. “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.”
