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. 2023 Jan 27;13(1):1520.
doi: 10.1038/s41598-023-28844-1.

Direct lysis of 3D cell cultures for RT-qPCR gene expression quantification

Fien Gysens  1   2 , Lisa Ostyn  3 , Ellen Goeteyn  3 , Eva Blondeel  4 , Justine Nuyttens  1   2 , Olivier De Wever  4 , Eric de Bony  1   2 , Aurélie Crabbé  3 , Pieter Mestdagh  5   6
Affiliations

Direct lysis of 3D cell cultures for RT-qPCR gene expression quantification

Fien Gysens et al. Sci Rep. .

Abstract

In vitro cell culture experiments are widely used to study cellular behavior in most biological research fields. Except for suspension cells, most human cell types are cultured as adherent monolayers on a plastic surface. While technically convenient, monolayer cultures can suffer from limitations in terms of physiological relevance, as their resemblance to complex in vivo tissue structures is limited. To address these limitations, three-dimensional (3D) cell culture systems have gained increased interest as they mimic key structural and functional properties of their in vivo tissue counterparts. Nevertheless, protocols established on monolayer cell cultures may require adjustments if they are to be applied to 3D cell cultures. As gene expression quantification is an essential part of many in vitro experiments, we evaluated and optimized a direct cell lysis, reverse transcription and qPCR protocol applicable for 3D cell cultures. The newly developed protocol wherein gene expression is determined directly from crude cell lysates showed improved cell lysis compared to the standard protocol, accurate gene expression quantification, hereby avoiding time-consuming cell harvesting and RNA extraction.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Cq values for five housekeeping genes (TBP, UBC, SDHA, HPRT1, and B2M), measured in two cell lines (BEAS-2B (A) and A549 (B)) on RNA extracted from 3D cell cultures (grey), lysates generated using the Singleshot protocol version 1 (red) or lysates generated using the Singleshot protocol version 2 (blue). Cq values for five additional genes (IL8, NFkB, NEAT1, MALAT1 and TGFBR1), measured in two cell lines (BEAS-2B (C) and A549 (D)) on RNA extracted from 3D cell cultures (grey), lysates generated using the Singleshot protocol version 1 (red) or lysates generated using the Singleshot protocol version 2 (blue). Data are presented as mean ± SE with *P < 0.05; **P < 0.01 and ***P < 0.001 (n = 3).
Figure 2
Figure 2
Correlation between Cq values from extracted RNA and lysate for lysis protocol version 2 in two different cell lines (BEAS-2B (A) and A549 (B)).
Figure 3
Figure 3
Correlation between delta Cq values (A549-BEAS-2B) from extracted RNA and lysate for both the version 1 and 2 lysis protocols in two different cell lines (BEAS-2B (A) and A549 (B)).
Figure 4
Figure 4
Cq values for 10 genes measured in three cell lines (A549 (A), HTC116 (B) and PANC1 (C)) on RNA extractions (grey) and on lysates generated using the Singleshot protocol version 2 (blue) or lysates generated using the Singleshot protocol version 2 and an additional Trypsin step (red). Data are presented as mean ± SE with *P < 0.05; **P < 0.01 and ***P < 0.001 (n = 3). Delta Cq values of house keeping genes between cell lines for 8 and 16 spheroids (D).
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