TY - JOUR
T1 - A systematic comparison of protocols for recovery of high-quality rna from human islets extracted by laser capture microdissection
AU - Cefalo, C. M. A.
AU - Mezza, Teresa
AU - Giaccari, Andrea
AU - Kulkarni, R. N.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - Human islets
KW - Laser capture microdissection
KW - RNA extraction
KW - Human islets
KW - Laser capture microdissection
KW - RNA extraction
UR - https://publicatt.unicatt.it/handle/10807/179349
UR - https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=85104532838&origin=inward
UR - https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85104532838&origin=inward
U2 - 10.3390/biom11050625
DO - 10.3390/biom11050625
M3 - Article
SN - 2218-273X
VL - 11
SP - 625
EP - 632
JO - Biomolecules
JF - Biomolecules
IS - 5
ER -