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.

Department of Pharmacology 1st Faculty of Medicine Charles University Praha Czech Republic

Department of Pharmacology General University Hospital Praha Czech Republic

Department of Physiology Faculty of Science Charles University Praha Czech Republic

Department of Rheumatology Institute of Rheumatology Praha Czech Republic

Faculty Transfusion Center General University Hospital Praha Czech Republic

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