An optimized QIAzol-based protocol for simultaneous miRNA, RNA, and protein isolation from precision-cut lung slices (PCLS)
Abstract Background Precision-cut lung slices (PCLS) are ex vivo models with preserved lung cell populations and maintained tissue architecture. PCLS are, therefore, a powerful tool in respiratory research to study molecular mechanisms that closely reflect whole tissue biology. High-quality RNA and...
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BMC
2024-11-01
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| Series: | Respiratory Research |
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| Online Access: | https://doi.org/10.1186/s12931-024-03026-3 |
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| author | Wojciech Langwiński Joanna Nowakowska Kosma Sakrajda Kamil Ziarniak Zuzanna Stachowiak Maria Kachel Beata Narożna Aleksandra Szczepankiewicz |
| author_facet | Wojciech Langwiński Joanna Nowakowska Kosma Sakrajda Kamil Ziarniak Zuzanna Stachowiak Maria Kachel Beata Narożna Aleksandra Szczepankiewicz |
| author_sort | Wojciech Langwiński |
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| description | Abstract Background Precision-cut lung slices (PCLS) are ex vivo models with preserved lung cell populations and maintained tissue architecture. PCLS are, therefore, a powerful tool in respiratory research to study molecular mechanisms that closely reflect whole tissue biology. High-quality RNA and protein extraction from PCLS is, however, challenging as agarose significantly interferes with the yield and purity of extracted material. The present study aimed to optimize QIAzol-based isolation protocol for high-yield and quality RNA, miRNA, and protein extraction from PCLS. Materials and methods PCLS were prepared from 10 to 15-week-old Wistar rats and cultured for 7 days in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 0.1% FBS, penicillin, and streptomycin. LDH release to PCLS culture media was measured to determine cellular cytotoxicity. To select the optimal miRNA/RNA isolation protocol, we tested two different times (10 min, 2 h) and temperatures (room temperature, 4 °C, and −20 °C) of precipitation with isopropanol. Finally, we also assessed isolation with GHCL (guanidinium hydrochloride) extraction buffer. To select the optimal protein isolation protocol, we tested protein precipitation for 10 min at room temperatures (21 ± 1 °C) with 1.5 volumes of isopropanol and 3 volumes of acetone per 1 volume of phenol-ethanol supernatant. Additionally, we also tested protein precipitation for 3 h at −20 °C with 3, 5, and 7 acetone volumes per 1 volume of phenol-ethanol supernatant. We also validated protein precipitation with back extraction buffer instead of 100% ethanol. To measure the general efficiency of the optimized QIAZ-4 protocol, we used native rat lungs. PCLS for the ex vivo model of allergic inflammation were treated with IL-13 at a concentration of 80 ng/ml. Results Standard QIAzol isolation protocol provided RNA, miRNA, and protein with low yield and poor quality. We found that 2-h isopropanol precipitation at 4 °C with a high concentration of salts significantly increased the yield and quality of extracted RNA and miRNA and provided acceptable qPCR efficiency (between 90 and 110%). Surprisingly, 2-h isopropanol precipitation at −20 °C significantly increased qPCR efficiency above the acceptable range (average efficiency: 120.4%). As for protein extraction, we found that 3-h acetone precipitation at −20 °C provided the highest yield with linear protein detection on Westen Blot. Optimized QIAZ-4 provided significantly higher miRNA and RNA yield compared to standard QIAzol protocols. We also found a significantly increased expression of Eotaxin-1 in PCLS treated with IL-13 as compared to the untreated controls. Conclusions In our study, we described a simple QIAzol-based method for the simultaneous isolation of RNA, miRNA, and protein from PCLS. |
| format | Article |
| id | doaj-art-9aa03314b2ae4d18b4fc1e63f0cf4264 |
| institution | Kabale University |
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| language | English |
| publishDate | 2024-11-01 |
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| series | Respiratory Research |
| spelling | doaj-art-9aa03314b2ae4d18b4fc1e63f0cf42642024-12-01T12:41:20ZengBMCRespiratory Research1465-993X2024-11-0125111610.1186/s12931-024-03026-3An optimized QIAzol-based protocol for simultaneous miRNA, RNA, and protein isolation from precision-cut lung slices (PCLS)Wojciech Langwiński0Joanna Nowakowska1Kosma Sakrajda2Kamil Ziarniak3Zuzanna Stachowiak4Maria Kachel5Beata Narożna6Aleksandra Szczepankiewicz7Molecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical SciencesMolecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical SciencesMolecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical SciencesMolecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical SciencesMolecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical SciencesMolecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical SciencesMolecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical SciencesMolecular and Cell Biology Unit, Department of Pediatric Pulmonology, Allergy and Clinical Immunology, Poznan University of Medical SciencesAbstract Background Precision-cut lung slices (PCLS) are ex vivo models with preserved lung cell populations and maintained tissue architecture. PCLS are, therefore, a powerful tool in respiratory research to study molecular mechanisms that closely reflect whole tissue biology. High-quality RNA and protein extraction from PCLS is, however, challenging as agarose significantly interferes with the yield and purity of extracted material. The present study aimed to optimize QIAzol-based isolation protocol for high-yield and quality RNA, miRNA, and protein extraction from PCLS. Materials and methods PCLS were prepared from 10 to 15-week-old Wistar rats and cultured for 7 days in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 0.1% FBS, penicillin, and streptomycin. LDH release to PCLS culture media was measured to determine cellular cytotoxicity. To select the optimal miRNA/RNA isolation protocol, we tested two different times (10 min, 2 h) and temperatures (room temperature, 4 °C, and −20 °C) of precipitation with isopropanol. Finally, we also assessed isolation with GHCL (guanidinium hydrochloride) extraction buffer. To select the optimal protein isolation protocol, we tested protein precipitation for 10 min at room temperatures (21 ± 1 °C) with 1.5 volumes of isopropanol and 3 volumes of acetone per 1 volume of phenol-ethanol supernatant. Additionally, we also tested protein precipitation for 3 h at −20 °C with 3, 5, and 7 acetone volumes per 1 volume of phenol-ethanol supernatant. We also validated protein precipitation with back extraction buffer instead of 100% ethanol. To measure the general efficiency of the optimized QIAZ-4 protocol, we used native rat lungs. PCLS for the ex vivo model of allergic inflammation were treated with IL-13 at a concentration of 80 ng/ml. Results Standard QIAzol isolation protocol provided RNA, miRNA, and protein with low yield and poor quality. We found that 2-h isopropanol precipitation at 4 °C with a high concentration of salts significantly increased the yield and quality of extracted RNA and miRNA and provided acceptable qPCR efficiency (between 90 and 110%). Surprisingly, 2-h isopropanol precipitation at −20 °C significantly increased qPCR efficiency above the acceptable range (average efficiency: 120.4%). As for protein extraction, we found that 3-h acetone precipitation at −20 °C provided the highest yield with linear protein detection on Westen Blot. Optimized QIAZ-4 provided significantly higher miRNA and RNA yield compared to standard QIAzol protocols. We also found a significantly increased expression of Eotaxin-1 in PCLS treated with IL-13 as compared to the untreated controls. Conclusions In our study, we described a simple QIAzol-based method for the simultaneous isolation of RNA, miRNA, and protein from PCLS.https://doi.org/10.1186/s12931-024-03026-3Extraction yieldQIAzolmiRNARNAProtein |
| spellingShingle | Wojciech Langwiński Joanna Nowakowska Kosma Sakrajda Kamil Ziarniak Zuzanna Stachowiak Maria Kachel Beata Narożna Aleksandra Szczepankiewicz An optimized QIAzol-based protocol for simultaneous miRNA, RNA, and protein isolation from precision-cut lung slices (PCLS) Respiratory Research Extraction yield QIAzol miRNA RNA Protein |
| title | An optimized QIAzol-based protocol for simultaneous miRNA, RNA, and protein isolation from precision-cut lung slices (PCLS) |
| title_full | An optimized QIAzol-based protocol for simultaneous miRNA, RNA, and protein isolation from precision-cut lung slices (PCLS) |
| title_fullStr | An optimized QIAzol-based protocol for simultaneous miRNA, RNA, and protein isolation from precision-cut lung slices (PCLS) |
| title_full_unstemmed | An optimized QIAzol-based protocol for simultaneous miRNA, RNA, and protein isolation from precision-cut lung slices (PCLS) |
| title_short | An optimized QIAzol-based protocol for simultaneous miRNA, RNA, and protein isolation from precision-cut lung slices (PCLS) |
| title_sort | optimized qiazol based protocol for simultaneous mirna rna and protein isolation from precision cut lung slices pcls |
| topic | Extraction yield QIAzol miRNA RNA Protein |
| url | https://doi.org/10.1186/s12931-024-03026-3 |
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