A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection

Cefalo, Chiara M. A.; Mezza, Teresa; Giaccari, Andrea; Kulkarni, Rohit N.

doi:10.3390/biom11050625

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A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection

¹

Islet Cell Biology & Regenerative Medicine, Joslin Diabetes Center, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA 02215, USA

²

Dipartimento di Scienze Mediche e Chirurgiche, Centro per le Malattie Endocrino-Metaboliche, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, 00168 Rome, Italy

³

Dipartimento di Medicina e Chirurgia Traslazionale, Università Cattolica del Sacro Cuore, 00168 Rome, Italy

^*

Authors to whom correspondence should be addressed.

Biomolecules 2021, 11(5), 625; https://doi.org/10.3390/biom11050625

Submission received: 2 April 2021 / Revised: 20 April 2021 / Accepted: 20 April 2021 / Published: 22 April 2021

(This article belongs to the Special Issue Pancreatic Islets of Langerhans: Not Only Beta-Cells)

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Abstract

The isolation of high-quality RNA from endocrine pancreas sections represents a considerable challenge largely due to the high ribonuclease levels. Laser capture microdissection (LCM) of mammalian islets, in association with RNA extraction protocols, has emerged as a feasible approach to characterizing their genetic and proteomic profiles. However, a validated protocol to obtain high-quality RNA from LCM-derived human pancreas specimens that is appropriate for next-generation sequencing analysis is still lacking. In this study, we applied four methods (Picopure extraction kit, Qiazol protocol, Qiazol + Clean-up kit, and RNeasy Microkit + Carrier) to extract RNA from human islets obtained from both non-diabetic individuals and patients with type 2 diabetes who had undergone partial pancreatectomy, as well as handpicked islets from both non-diabetic and diabetic organ donors. The yield and purity of total RNA were determined by 260/280 absorbance using Nanodrop 100 and the RNA integrity number with a bioanalyzer. The results indicated that among the four methods, the RNeasy MicroKit + Carrier (Qiagen) provides the highest yield and purity.

Keywords: laser capture microdissection; RNA extraction; human islets

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MDPI and ACS Style

Cefalo, C.M.A.; Mezza, T.; Giaccari, A.; Kulkarni, R.N. A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection. Biomolecules 2021, 11, 625. https://doi.org/10.3390/biom11050625

AMA Style

Cefalo CMA, Mezza T, Giaccari A, Kulkarni RN. A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection. Biomolecules. 2021; 11(5):625. https://doi.org/10.3390/biom11050625

Chicago/Turabian Style

Cefalo, Chiara M. A., Teresa Mezza, Andrea Giaccari, and Rohit N. Kulkarni. 2021. "A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection" Biomolecules 11, no. 5: 625. https://doi.org/10.3390/biom11050625

APA Style

Cefalo, C. M. A., Mezza, T., Giaccari, A., & Kulkarni, R. N. (2021). A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection. Biomolecules, 11(5), 625. https://doi.org/10.3390/biom11050625

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A Systematic Comparison of Protocols for Recovery of High-Quality RNA from Human Islets Extracted by Laser Capture Microdissection

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