The isolation of high-quality RNA from endocrine pancreas sections represents a consider-able 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.

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 (5): 625-632. [doi:10.3390/biom11050625] [http://hdl.handle.net/10807/179349]

A systematic comparison of protocols for recovery of high-quality rna from human islets extracted by laser capture microdissection

Cefalo, C. M. A.
Primo
;
Mezza, T.;Giaccari, A.
Ultimo
;
2021

Abstract

The isolation of high-quality RNA from endocrine pancreas sections represents a consider-able 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.
Inglese
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 (5): 625-632. [doi:10.3390/biom11050625] [http://hdl.handle.net/10807/179349]
File in questo prodotto:
File Dimensione Formato  
biomolecules-11-00625-v2.pdf

accesso aperto

Tipologia file ?: Postprint (versione finale dell’autore successiva alla peer-review)
Licenza: Creative commons
Dimensione 10.42 MB
Formato Adobe PDF
10.42 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10807/179349
Citazioni
  • ???jsp.display-item.citation.pmc??? 3
  • Scopus 4
  • ???jsp.display-item.citation.isi??? 4
social impact