1. Academic Validation
  2. Simple, streamlined, cost-effective cDNA synthesis method from cell cultures

Simple, streamlined, cost-effective cDNA synthesis method from cell cultures

  • Open Biol. 2025 Mar;15(3):240226. doi: 10.1098/rsob.240226.
Daniel Stránský 1 2 Monika Šteigerová 1 2 Markéta Kuklová 3 Veronika Hanzíková 4 Nikolina Canová 1 Jiří Novotný 5 Ladislav Šenolt 3 Ondřej Slanař 1 2
Affiliations

Affiliations

  • 1 Department of Pharmacology, First Faculty of Medicine, Charles University, Praha, Czech Republic.
  • 2 Department of Pharmacology, General University Hospital, Praha, Czech Republic.
  • 3 Department of Rheumatology, Institute of Rheumatology, Praha, Czech Republic.
  • 4 Faculty Transfusion Center, General University Hospital, Praha, Czech Republic.
  • 5 Department of Physiology, Faculty of Science, Charles University, Praha, Czech Republic.
Abstract

Applications like drug development need simple and streamlined methods to process samples from 96-well Cell Culture plates for gene expression measurements. Unfortunately, current options are expensive for such processing. Therefore, our aim was to develop a method that would allow streamlined analysis of mRNA from 96-well Cell Culture plates while being relatively cheap and simple. We developed a method based on the qPCR 'Cells-to-cDNA' approach and validated it against commercially available kits using the same approach or spin columns-based RNA purification. For this purpose, we conducted a series of comparisons of gene expression from peripheral blood mononuclear cells, SK-HEP-1 and U-87 cell cultures in 96-well plates. Our final method involved lysing cells with 25-100 µl solution of 0.5% SDS, 10 mM DTT, 1 mg ml-1 proteinase K dissolved in water, 1 h incubation at 50°C, followed by proteinase K inactivation at 90°C for 5 min and lysate neutralization with 1 : 1 dilution by 20% Tween 20 solution. Reverse transcription and qPCR were carried out using standard methods. This method showed a mean reduction of Ct ± s.d. value by 2.4 ± 1.3 compared with the 'Cells-to-cDNA' kit and by 1.4 ± 0.5 compared with the RNA purification kit with lower variability.

Keywords

RNA isolation; cell lysis; in vitro; mRNA; peripheral blood mononuclear cells; proteinase k; qPCR.

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