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A systematic comparison of in-house prepared transfection reagents in the delivery of mRNA or DNA to a wide range of cultured cells.

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Title: A systematic comparison of in-house prepared transfection reagents in the delivery of mRNA or DNA to a wide range of cultured cells.
Authors: Ojha, Ravi1, Timin, Emilia1, Szirovicza, Leonora1, Xu, Wujun2, Hepojoki, Jussi1,3 Jussi.hepojoki@helsinki.fi
Source: Journal of Biological Chemistry. Dec2025, Vol. 301 Issue 12, p1-16. 16p.
Subjects: Messenger RNA, DNA, Gene transfection, Cell culture, Cationic lipids, Genome editing, Chemical reagents, Cytotoxins
Abstract: Transfection is a fundamental molecular biology technique, enabling gene editing, protein expression, and vaccine development. However, transfection efficiency and cytotoxicity vary widely between reagent and cell type, necessitating optimization. Commercial reagents such as Lipofectamine 2000 and FuGENE HD are widely used for their high efficiency, but they are expensive and the efficiency can associate with cytotoxicity. In-house alternatives such as linear PEI (25 kDa and 40 kDa) and cationic lipids—1,2-di-O-octadecenyl-3-trimethylammonium propane and 1,2-dioleoyl-3-trimethylammonium-propane—combined with dioleoylphosphatidylethanolamine (DOPE) offer costeffective options, but their performance across diverse cell types and nucleic acid type (RNA or DNA) remains insufficiently characterized. This motivated us to systematically evaluate transfectionefficiency, cytotoxicity, andcomplex stability ofthese in-house reagents (DOPE: 1,2-dioleoyl-3-trimethylammoniumpropane and DOPE:N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride tested at molar ratios of 0.5:1, 1:1, and 2:1) over a broad range of reagent to nucleic acid ratios, using plasmid DNA and mRNA encoding mCherry. We performed transfections across 14 cell lines derived from human, monkey, frog, snake, and rodent tissues. We utilized automated fluorescence microscopy for quantifying transfection efficiency, luminescence-based viability assays for cytotoxicity, and studied complex stability during storage at 4 ◦C (0, 4, and 24 h) through transfection. Results revealed cell line-dependent differences in transfection efficiency and showed in-house cationic lipid formulations to have a high mRNA transfection efficiency with low cytotoxicity. Lipofectamine 2000 and PEI 40k formed the most stable DNA complexes, but with higher cytotoxicity. This study provides a comprehensive reference for selecting customizable, cost-effective transfection reagents for specific cell and nucleic acid types. [ABSTRACT FROM AUTHOR]
Copyright of Journal of Biological Chemistry is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
Database: Engineering Source
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Header DbId: egs
DbLabel: Engineering Source
An: 190653014
AccessLevel: 6
PubType: Academic Journal
PubTypeId: academicJournal
PreciseRelevancyScore: 0
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Items – Name: Title
  Label: Title
  Group: Ti
  Data: A systematic comparison of in-house prepared transfection reagents in the delivery of mRNA or DNA to a wide range of cultured cells.
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  Label: Authors
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  Data: <searchLink fieldCode="AR" term="%22Ojha%2C+Ravi%22">Ojha, Ravi</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Timin%2C+Emilia%22">Timin, Emilia</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Szirovicza%2C+Leonora%22">Szirovicza, Leonora</searchLink><relatesTo>1</relatesTo><br /><searchLink fieldCode="AR" term="%22Xu%2C+Wujun%22">Xu, Wujun</searchLink><relatesTo>2</relatesTo><br /><searchLink fieldCode="AR" term="%22Hepojoki%2C+Jussi%22">Hepojoki, Jussi</searchLink><relatesTo>1,3</relatesTo><i> Jussi.hepojoki@helsinki.fi</i>
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  Data: <searchLink fieldCode="JN" term="%22Journal+of+Biological+Chemistry%22">Journal of Biological Chemistry</searchLink>. Dec2025, Vol. 301 Issue 12, p1-16. 16p.
– Name: Subject
  Label: Subjects
  Group: Su
  Data: <searchLink fieldCode="DE" term="%22Messenger+RNA%22">Messenger RNA</searchLink><br /><searchLink fieldCode="DE" term="%22DNA%22">DNA</searchLink><br /><searchLink fieldCode="DE" term="%22Gene+transfection%22">Gene transfection</searchLink><br /><searchLink fieldCode="DE" term="%22Cell+culture%22">Cell culture</searchLink><br /><searchLink fieldCode="DE" term="%22Cationic+lipids%22">Cationic lipids</searchLink><br /><searchLink fieldCode="DE" term="%22Genome+editing%22">Genome editing</searchLink><br /><searchLink fieldCode="DE" term="%22Chemical+reagents%22">Chemical reagents</searchLink><br /><searchLink fieldCode="DE" term="%22Cytotoxins%22">Cytotoxins</searchLink>
– Name: Abstract
  Label: Abstract
  Group: Ab
  Data: Transfection is a fundamental molecular biology technique, enabling gene editing, protein expression, and vaccine development. However, transfection efficiency and cytotoxicity vary widely between reagent and cell type, necessitating optimization. Commercial reagents such as Lipofectamine 2000 and FuGENE HD are widely used for their high efficiency, but they are expensive and the efficiency can associate with cytotoxicity. In-house alternatives such as linear PEI (25 kDa and 40 kDa) and cationic lipids—1,2-di-O-octadecenyl-3-trimethylammonium propane and 1,2-dioleoyl-3-trimethylammonium-propane—combined with dioleoylphosphatidylethanolamine (DOPE) offer costeffective options, but their performance across diverse cell types and nucleic acid type (RNA or DNA) remains insufficiently characterized. This motivated us to systematically evaluate transfectionefficiency, cytotoxicity, andcomplex stability ofthese in-house reagents (DOPE: 1,2-dioleoyl-3-trimethylammoniumpropane and DOPE:N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride tested at molar ratios of 0.5:1, 1:1, and 2:1) over a broad range of reagent to nucleic acid ratios, using plasmid DNA and mRNA encoding mCherry. We performed transfections across 14 cell lines derived from human, monkey, frog, snake, and rodent tissues. We utilized automated fluorescence microscopy for quantifying transfection efficiency, luminescence-based viability assays for cytotoxicity, and studied complex stability during storage at 4 ◦C (0, 4, and 24 h) through transfection. Results revealed cell line-dependent differences in transfection efficiency and showed in-house cationic lipid formulations to have a high mRNA transfection efficiency with low cytotoxicity. Lipofectamine 2000 and PEI 40k formed the most stable DNA complexes, but with higher cytotoxicity. This study provides a comprehensive reference for selecting customizable, cost-effective transfection reagents for specific cell and nucleic acid types. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
  Group: Ab
  Data: <i>Copyright of Journal of Biological Chemistry is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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RecordInfo BibRecord:
  BibEntity:
    Identifiers:
      – Type: doi
        Value: 10.1016/j.jbc.2025.110742
    Languages:
      – Code: eng
        Text: English
    PhysicalDescription:
      Pagination:
        PageCount: 16
        StartPage: 1
    Subjects:
      – SubjectFull: Messenger RNA
        Type: general
      – SubjectFull: DNA
        Type: general
      – SubjectFull: Gene transfection
        Type: general
      – SubjectFull: Cell culture
        Type: general
      – SubjectFull: Cationic lipids
        Type: general
      – SubjectFull: Genome editing
        Type: general
      – SubjectFull: Chemical reagents
        Type: general
      – SubjectFull: Cytotoxins
        Type: general
    Titles:
      – TitleFull: A systematic comparison of in-house prepared transfection reagents in the delivery of mRNA or DNA to a wide range of cultured cells.
        Type: main
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            NameFull: Ojha, Ravi
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            NameFull: Timin, Emilia
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            NameFull: Szirovicza, Leonora
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            NameFull: Xu, Wujun
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            NameFull: Hepojoki, Jussi
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          Dates:
            – D: 01
              M: 12
              Text: Dec2025
              Type: published
              Y: 2025
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            – Type: issn-print
              Value: 00219258
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              Value: 301
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              Value: 12
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            – TitleFull: Journal of Biological Chemistry
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