Abstract Introduction Precision cut lung slices (PCLS) have emerged as a novel tool to study biology in living human lungs. PCLS involves agarose gel inflation to facilitate vibratome sectioning and maintenance of pulmonary microarchitecture. However, agarose carryover during RNA extraction reduce RNA integrity and yield.1 Here we assess whether dissolving agarose before RNA extraction or using a different RNA kit improvesRNA yield and integrity. Methods This study received approval from the Yale University Institutional Review Board. PCLS were generated as previously described. Briefly, human lungs were inflated with a 2% agarose solution. Inflated lungs were cored using 12 mm punch and sectioned in a Precisionary vibratome. 400 µm-thick sections were incubated in DMEM:F12K with 0.1% BSA and 1% Antibiotic-Antimycotic with or without fibrotic cocktail (FC). PCLS were harvested after 6 days. We used two RNA extraction methods (Qiagen miRNeasy or Plant Kit) and PCLS with agarose (Agarose+) or PCLS which agarose was dissolved using ZymoResearch RAD buffer (Agarose-) (Fig.1a). We monitored PCLS viability by Calcein (live) and Ethidium homodimer-1 (dead) staining, imaged in a LeicaStellaris Confocal microscope and quantified the fluorescence in ImageJ. We measured RNA quantity, quality and integrity in NanoDrop2000, Qubit2.0 and 2100Bioanalyzer. We measured gene expression by qPCR in a QuantStudio6Pro. Graphs and statistics were performed in GraphPad 9.5.0. Results Live-dead staining confirmed cells were viable during culture conditions (Fig.1b). The RNA extraction for conventional agarose-containing PCLS showed inconsistent measurements among quantification techniques, high presence of impurities (260/280=1.56±0.05; 260/230=0.12±0.02) and absence of intact rRNA (Fig. 1c,d,e,f). Agarose dissolution led to consistent RNA quantifications, reduced impurities (260/280=1.88±0.06; 260/230=1.33±0.45) and intact rRNA (RIN=9.07±0.47). While the Plant kit still showed inconsistent RNA quantity it showed reduced of impurities (260/280=1.75±0.06; 260/230=1.56±0.16) and improved RNA (RIN=5.95±0.93). GUSB and COL1A1 expressionmeasured in the RNA obtained from the Agarose dissolution and Plant kit weresignificantly higher, suggesting that transcript integrity was improved. (Fig.1g). Conclusions The presence of agarose reducesthe quality, integrity and yield of RNA from PCLS using usual extractionmethods. Of the methods tested dissolution of agarose consistentlyimproves both RNA yield and integrity whereas the Plant Kit improves but isless consistent References: 1 DOI:10.1186/s13104-015-1627-5 This abstract is funded by: European Commission H2020-MSC-IF (101033565)
Munoz et al. (Fri,) studied this question.