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RNA and protein isolation from precision-cut lung slices (PCLS) using QIAzol Lysis Reagent

Version 1.0

Langwiński, Wojciech; Nowakowska-Lewicka, Joanna; Sakrajda, Kosma; Ziarniak, Kamil; Stachowiak, Zuzanna; Kachel, Maria; Narozna, Beata; Szczepankiewicz, Aleksandra, 2026, "RNA and protein isolation from precision-cut lung slices (PCLS) using QIAzol Lysis Reagent", https://doi.org/10.18150/7AP5YA, RepOD, V1

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Description

Precision-cut lung slices (PCLS) are ex vivo models generated by sectioning agarose-inflated lungs into slices of uniform thickness. Because PCLS preserve all lung cell populations along with native tissue architecture, they represent a powerful tool in respiratory research for studying molecular mechanisms that closely reflect whole-tissue biology.

However, the extraction of high-quality RNA from PCLS remains challenging, as residual agarose significantly interferes with both the yield and purity of isolated material. Agarose contamination impairs downstream molecular biology applications, including reverse transcription and quantitative real-time PCR, as well as high-throughput approaches such as next-generation sequencing and microarray analyses, thereby limiting the applicability of PCLS-based models.

The aim of this study was to optimize a QIAzol-based protocol for the simultaneous isolation of intact RNA and protein from PCLS. To this end, we evaluated several protocol variants:

·        QIAZ-1: Standard QIAzol protocol

·        QIAZ-2: RNA precipitation for 2 h at −20°C

·        QIAZ-3: RNA precipitation for 2 h at +4°C using 200 µL QIAzol

·        QIAZ-4: RNA precipitation for 2 h at +4°C using 100 µL QIAzol

·        GHCL-1: Guanidinium hydrochloride-based extraction buffer with additional NaCl supplementation

·        GHCL-2: Guanidinium hydrochloride-based extraction buffer without NaCl supplementation

RNA yield and purity (A260/230 ratio) were assessed using a NanoDrop spectrophotometer, while RNA integrity was determined by RNA Integrity Number (RIN) using a TapeStation system. miRNA concentration was quantified using a fluorometric assay. In addition, lactate dehydrogenase (LDH) activity was measured..

To evaluate suitability for downstream applications, we assessed amplification efficiency of GAPDH (mRNA) and rno-miR-223-3p (miRNA) using real-time PCR.

Among the tested protocols, QIAZ-4 showed the most promising performance. For this protocol, protein quantification linearity was evaluated using FKBP5 in rat samples across a range of protein inputs (1.25, 2.5, 5, and 10 µg). A strong linear relationship between normalized signal intensity and protein load was observed (R² = 0.961), indicating high reliability for downstream protein analysis.

Subject
Medicine, Health and Life Sciences
Keyword

PCLS, lungs, miRNA, rno-miR-223-3p

Related Publication

Langwiński W, Nowakowska J, Sakrajda K, Ziarniak K, Stachowiak Z, Kachel M, Narożna B, Szczepankiewicz A. An optimized QIAzol-based protocol for simultaneous miRNA, RNA, and protein isolation from precision-cut lung slices (PCLS). Respir Res. 2024 Nov 30;25(1):422. doi: 10.1186/s12931-024-03026-3. PMID: 39616338; PMCID: PMC11608482 https://doi.org/10.1186/s12931-024-03026-3 doi: 10.1186/s12931-024-03026-3

CC BY - Creative Commons Attribution 4.0 CC BY - Creative Commons Attribution 4.0

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1 to 5 of 5 Files
LDH.tab
Tabular Data - Original format: MS Excel (XLSX) - 266 B - Mar 20, 2026 - 0 Downloads
License: CC BY - Creative Commons Attribution 4.0
2 Variables, 18 Observations - UNF:6:uPXi9TUjAUctxQ7vbHna+Q==
LDH values were expressed as optical density (OD).
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protein_concentration.tab
Tabular Data - Original format: MS Excel (XLSX) - 569 B - Mar 20, 2026 - 0 Downloads
License: CC BY - Creative Commons Attribution 4.0
2 Variables, 21 Observations - UNF:6:YNZygYS8mAdrUak99AEZBw==
Protein concentration (µg) was measured using the Pierce BCA assay.
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QIAZ_4_protein_linearity.tab
Tabular Data - Original format: MS Excel (XLSX) - 197 B - Mar 20, 2026 - 0 Downloads
License: CC BY - Creative Commons Attribution 4.0
5 Variables, 4 Observations - UNF:6:Bn/41RlEIh3dn3fP4L0BHw==
Protein linearity was evaluated by Western blot across increasing protein loads.
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qPCR_effiiciency.tab
Tabular Data - Original format: MS Excel (XLSX) - 158 B - Mar 20, 2026 - 0 Downloads
License: CC BY - Creative Commons Attribution 4.0
4 Variables, 7 Observations - UNF:6:tNaX9pJe4s4I3GAJHbrvTQ==
qPCR effiiciency of different protocols.
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RNA.tab
Tabular Data - Original format: MS Excel (XLSX) - 906 B - Mar 20, 2026 - 0 Downloads
License: CC BY - Creative Commons Attribution 4.0
6 Variables, 18 Observations - UNF:6:I1zTWOaWg30WLeqw0r5SSg==
RNA quality parameters were assessed using a NanoDrop spectrophotometer, fluorometric quantification, and a TapeStation system.
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