Miniaturization of gene transfection assays in 384- and 1536-well microplates

Jing Li; Samuel T Crowley; Jason Duskey; Sanjib Khargharia; Meng Wu; Kevin G Rice

doi:10.1016/j.ab.2014.10.001

Back

Miniaturization of gene transfection assays in 384- and 1536-well microplates

Journal article

Open access

Peer reviewed

Miniaturization of gene transfection assays in 384- and 1536-well microplates

Jing Li, Samuel T Crowley, Jason Duskey, Sanjib Khargharia, Meng Wu and Kevin G Rice

Analytical biochemistry, Vol.470, pp.14-21

02/01/2015

DOI: 10.1016/j.ab.2014.10.001

PMCID: PMC4601643

PMID: 25448623

Files and links (1)

url

http://hdl.handle.net/11380/1205678View

Open Access

Abstract

The miniaturization of gene transfer assays to either 384- or 1536-well plates greatly economizes the expense and allows much higher throughput when transfecting immortalized and primary cells compared with more conventional 96-well assays. To validate the approach, luciferase and green fluorescent protein (GFP) reporter gene transfer assays were developed to determine the influence of cell seeding number, transfection reagent to DNA ratios, transfection time, DNA dose, and luciferin dose on linearity and sensitivity. HepG2, CHO, and NIH 3T3 cells were transfected with polyethylenimine (PEI)–DNA in both 384- and 1536-well plates. The results established optimal transfection parameters in 384-well plates in a total assay volume of 35μl and in 1536-well plates in a total assay volume of 8μl. A luciferase assay performed in 384-well plates produced a Z′ score of 0.53, making it acceptable for high-throughput screening. Primary hepatocytes were harvested from mouse liver and transfected with PEI DNA and calcium phosphate DNA nanoparticles in 384-well plates. Optimal transfection of primary hepatocytes was achieved on as few as 250cellsperwell in 384-well plates, with CaPO4 proving to be 10-fold more potent than PEI.

1536-well

Gene delivery

Luciferase assay

Gene transfection

384-well

Mammalian cell culture

Details

Title: Subtitle: Miniaturization of gene transfection assays in 384- and 1536-well microplates
Creators: Jing Li - University of Iowa
Samuel T Crowley - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA
Jason Duskey - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA
Sanjib Khargharia - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA
Meng Wu - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA
Kevin G Rice - Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA 52242, USA
Resource Type: Journal article
Publication Details: Analytical biochemistry, Vol.470, pp.14-21
DOI: 10.1016/j.ab.2014.10.001
PMID: 25448623
PMCID: PMC4601643
NLM abbreviation: Anal Biochem
ISSN: 0003-2697
eISSN: 1096-0309
Publisher: Elsevier Inc
Grant note: DOI: 10.13039/100000002, name: National Institutes of Health, award: GM097093, T32GM008365; DOI: 10.13039/100005496, name: American Foundation for Pharmaceutical Education; name: University of Iowa High Throughput Screening Facility, award: S10 RR029274-01
Language: English
Date published: 02/01/2015
Academic Unit: Pharmacy; Core Research Facilities; Pharmaceutical Sciences and Experimental Therapeutics; Craniofacial Anomalies Research Center; Biochemistry and Molecular Biology; Medicinal and Natural Products Chemistry
Record Identifier: 9984065317702771

Metrics

11 Record Views

14 Times Cited - Web of Science