Modified RNA Extraction Methods to Eliminate Agarose Impurities in Human Precision-Cut Lung Slices

Abstract

Precision-cut lung slices (PCLS) have emerged as a powerful tool for studying the biology of viable human lung tissue. However, the presence of agarose impurities compromises RNA yield and integrity during extraction. We tested whether using an alternative Plant kit extraction to wash agarose impurities or pre-dissolution of agarose from PCLS by immersion in a dissolving buffer for routine RNA isolation in gel electrophoresis would improve RNA quantity, quality, and integrity. Our results show that RNA quantity and integrity are highly compromised when using a conventional method of RNA extraction. The Plant kit and dissolution of agarose efficiently removed agarose impurities and increased the RNA quantity from < 0.028 µg/PCLS (below the detection limit) to 0.42 ± 0.11 and 0.65 ± 0.17 µg/PCLS (measured by the Qubit) and integrity number from 2.27 ± 0.87 to 6.60 ± 0.59 and 9.13 ± 0.39 (measured by the Bioanalyzer), respectively. The presence of phenol impurities in conventional and Plant kit extractions led to an overestimation of the RNA quantity and quality using the NanoDrop. The Plant kit and agarose dissolution showed a significant transcription amplification increase of housekeeping GUSB (p < 0.0001) and COL1A1 (p < 0.05) by qPCR, validating these methods over conventional extraction. We encourage laboratories applying PCLS experimentation to implement alternative methods that efficiently remove agarose impurities during RNA extraction, as well as to rely on sensitive RNA quantitative techniques, such as the Qubit and Bioanalyzer, for quantification and integrity assessment.