Establishment of lipofection for studying miRNA function in human adipocytes.
miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNAs into these cells. To achieve this, we compared the efficiencies of three transfection agents, Lipofectamine 2000, ScreenFect A and BPEI 1.2 k in delivering fluorescent-labelled siRNA into human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes and adipocytes. Downregulation of a specific target gene in response to miRNA mimic overexpression was assayed in SGBS cells and also in ex vivo differentiated primary human adipocytes. Our results demonstrated that while all three transfection agents were able to internalize the oligos, only lipofection resulted in the efficient downregulation of a specific target gene both in SGBS cells and in primary human adipocytes. Lipofectamine 2000 outperformed ScreenFect A in preadipocytes, but in adipocytes the two reagents gave comparable results making ScreenFect A a notable new alternative for the gold standard Lipofectamine 2000.
Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany.
Eveliina
E
Enlund
Institute of Applied Biotechnology, University of Applied Sciences Biberach, Biberach, Germany.
Simon
S
Fischer
Institute of Applied Biotechnology, University of Applied Sciences Biberach, Biberach, Germany.
René
R
Handrick
Institute of Applied Biotechnology, University of Applied Sciences Biberach, Biberach, Germany.
Kerstin
K
Otte
Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany.
Klaus-Michael
KM
Debatin
Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany.
Martin
M
Wabitsch
Division of Pediatric Endocrinology and Diabetes, Department of Pediatrics and Adolescent Medicine, Ulm University Medical Center, Ulm, Germany.
Pamela
P
Fischer-Posovszky
D017667:Adipocytes / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D000328:Adult; D001145:Arrhythmias, Cardiac / Q000473:pathology; D002454:Cell Differentiation; D004337:Drug Carriers / Q000737:chemistry*; D005260:Female; D040181:Genetic Diseases, X-Linked / Q000473:pathology; D005877:Gigantism / Q000473:pathology; D006330:Heart Defects, Congenital / Q000473:pathology; D006801:Humans; D008607:Intellectual Disability / Q000473:pathology; D008055:Lipids / Q000737:chemistry*; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D004337:Drug Carriers; D008055:Lipids; C086724:Lipofectamine; D035683:MicroRNAs