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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Singh KP, Miaskowski C, Dhruva AA, Flowers E, Kober KM. 2018. Mechanisms and measurement of changes in gene expression. Biol. Res. Nurs. 20, 369–382. (10.1177/1099800418772161) PubMed DOI PMC

Yang T, Zhang M, Zhang N. 2022. Modified northern blot protocol for easy detection of mRNAs in total RNA using radiolabeled probes. BMC Genom. 23, 66. (10.1186/s12864-021-08275-w) PubMed DOI PMC

Jaksik R, Iwanaszko M, Rzeszowska-Wolny J, Kimmel M. 2015. Microarray experiments and factors which affect their reliability. Biol. Direct 10, 46. (10.1186/s13062-015-0077-2) PubMed DOI PMC

Martin SAM, Dehler CE, Król E. 2016. Transcriptomic responses in the fish intestine. Dev. Comp. Immunol. 64, 103–117. (10.1016/j.dci.2016.03.014) PubMed DOI

Bender AT, Sullivan BP, Lillis L, Posner JD. 2020. Enzymatic and chemical-based methods to inactivate endogenous blood ribonucleases for nucleic acid diagnostics. J. Mol. Diagn. 22, 1030–1040. (10.1016/j.jmoldx.2020.04.211) PubMed DOI PMC

Goldenberger D, Perschil I, Ritzler M, Altwegg M. 1995. A simple ‘universal’ DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. PCR Methods Appl. 4, 368–370. (10.1101/gr.4.6.368) PubMed DOI

Shatzkes K, Teferedegne B, Murata H. 2014. A simple, inexpensive method for preparing cell lysates suitable for downstream reverse transcription quantitative PCR. Sci. Rep. 4, 4659. (10.1038/srep04659) PubMed DOI PMC

Le AV, Huang D, Blick T, Thompson EW, Dobrovic A. 2015. An optimised direct lysis method for gene expression studies on low cell numbers. Sci. Rep. 5, 12859. (10.1038/srep12859) PubMed DOI PMC

Svec D, Andersson D, Pekny M, Sjöback R, Kubista M, Ståhlberg A. 2013. Direct cell lysis for single-cell gene expression profiling. Front. Oncol. 3, 274. (10.3389/fonc.2013.00274) PubMed DOI PMC

Rodríguez A, Duyvejonck H, Van Belleghem JD, Gryp T, Van Simaey L, Vermeulen S, Van Mechelen E, Vaneechoutte M. 2020. Comparison of procedures for RNA-extraction from peripheral blood mononuclear cells. PLoS One 15, e0229423. (10.1371/journal.pone.0229423) PubMed DOI PMC

Kunde DA, Yingchoncharoen J, Jurković S, Geraghty DP. 2018. TRPV1 mediates capsaicin-stimulated metabolic activity but not cell death or inhibition of interleukin-1β release in human THP-1 monocytes. Toxicol. Appl. Pharmacol. 360, 9–17. (10.1016/j.taap.2018.09.025) PubMed DOI

Bessler H, Djaldetti M. 2017. Capsaicin modulates the immune cross talk between human mononuclears and cells from two colon carcinoma lines. Nutr. Cancer 69, 14–20. (10.1080/01635581.2017.1247893) PubMed DOI

Erin N, Akman M. 2021. Effects of in-vitro modulation of TRPV1 activity on immune response of mice bearing metastatic breast carcinoma: enhanced inflammatory response may hinder therapeutic potentials of TRPV1 agonists. Life Sci. 287, 120115. (10.1016/j.lfs.2021.120115) PubMed DOI

Stránský D, Šteigerová M, Kuklová M, Hanzíková V, Canová K, Slanař O. 2024. Simple, high-throughput, cost-effective cDNA synthesis method from cell cultures. OSF. See https://osf.io/39qnh/?view_only=82e8a5f7d2b44058b6975cb5cb52539e. PubMed

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