Issue 33, 2022

Role of detergents and nuclease inhibitors in the extraction of RNA from eukaryotic cells in complex matrices

Abstract

The potential for liquid biopsy samples to be used in place of more invasive tissue biopsies has become increasingly revalent as it has been found that nucleic acids (NAs) present in the blood of cancer patients originate from tumors. Nanomagnetic extraction has proven to be a highly effective means to rapidly prepare NA from clinical samples for molecular diagnostics. In this article, the lysis reaction used to extract RNA from the human epithelial melanoma cells have been optimized using silica coated superparamagnetic nanoparticles (SPM NP). The lysis buffer (LB) is composed of several agents that denature cells, i.e., surfactant and guanidinium isothiocyanate (GITC), and agents that inhibit the degradation of circulated nucleic acids (cfNAs). The surfactant Triton X-100 has been widely used in LB but has been placed on the European Union REACH list. We have compared the qRT-PCR sensitivity resulting from LBs composed of Triton X-100 to several sustainable surfactants, i.e., Tergitol 15-S-7, Tergitol 15-S-9 and Tween-20. Surprisingly, the inclusion of these surfactants in the LB was not found to significantly improve cell lysis, and subsequently the sensitivity of qRT-PCR. The role of the sample matrix was also examined by performing extractions from solutions containing up to 30 mg mL−1 serum albumin. The qRT-PCR sensitivity was found to decrease as the concentration of this protein was increased; however, this was linked to an increased RNase activity and not the concentration of the protein itself. These results lead us to recommend a reformulation of LB for clinical samples, and to conclude that sensitive qRT-PCR RNA analysis can be performed in serum with the timely addition of an RNase inhibitor.

Graphical abstract: Role of detergents and nuclease inhibitors in the extraction of RNA from eukaryotic cells in complex matrices

Supplementary files

Article information

Article type
Paper
Submitted
23 May 2022
Accepted
31 Jul 2022
First published
09 Aug 2022
This article is Open Access
Creative Commons BY-NC license

Nanoscale, 2022,14, 12153-12161

Role of detergents and nuclease inhibitors in the extraction of RNA from eukaryotic cells in complex matrices

C. Holohan, N. Feely, P. Li, G. Curran and G. U. Lee, Nanoscale, 2022, 14, 12153 DOI: 10.1039/D2NR02850F

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