Elsevier

Talanta

Volume 200, 1 August 2019, Pages 51-56
Talanta

PCR-free and chemistry-based technology for miR-21 rapid detection directly from tumour cells

https://doi.org/10.1016/j.talanta.2019.03.039Get rights and content

Highlights

  • Chem-NAT based protocol to count miRNA molecules per cell with single base resolution directly from cell lysates.

  • Chem-NAT implementation for miRNA quantification with standard FACS and fluorescence microplate readers.

  • Three and half hours protocol from cell pellet to result which do not require nucleic acid amplification steps.

  • Quantification of miR-21 molecules presented in tumour cells MDA-MB-468 and H1975.

Abstract

miRNAs are well known for being implicated in a myriad of biological situations, including those related to serious diseases. Amongst miRNAs, miRNA-21 has the spotlight as it is reported to be up-regulated in multiple severe pathological conditions, being its quantification a key point in medicine. To date, most of the techniques for miRNA quantification have shown to be less effective than expected; thus, we herein present a novel, rapid, cost-effective, robust and PCR-free approach, based on dynamic chemistry, for the identification and quantification of miRNA directly from tumour cells using both FACS and a fluorescent microplate. This dynamic chemistry novel application involves bead based reagents and allows quantifying the number of miR-21 molecules presented in MDA-MB-468 and H1975 tumour cells.

Introduction

MicroRNAs (miRNAs) are small non-coding RNAs of 18–24 nucleotides in length that regulate gene expression by directly interacting with the 3′ untranslated region (UTR) of a target gene. This interaction leads to degradation and/or translational repression of that gene [1], [2], [3], [4]. miRNAs are implicated in the regulation of cell growth, differentiation, and apoptosis (and their deregulation is associated with multiple serious diseases [4].

Since the discovery of miRNAs, miR-21 has become one of the most studied and cited miRNAs, as it has been reported to be up-regulated in a lot of pathological conditions, such as glioblastoma, pancreatic cancer, breast cancer, lung cancer, and colon cancer. Besides, miR-21 up-regulation is directly related to cell proliferation, migration and invasion, and to the generation of chemoresistance in lung cancer [5], in breast cancer [5] and in ovarian cancer [6]. For that, it is considered an OncomiR [7]. Thus, miR-21 has emerged as one of the miRNA most frequently associated with poor outcome in cancer, being considered as a very promising therapeutic target for cancer [8], [9]. As a matter of fact, CRISPR/CAS9 based gene therapies targeting miR-21 are being developed nowadays [6], [7].

To date, miRNA analysis is mostly done by standard RT-qPCR. However, these tools are not particularly suitable for detecting small RNA species, since they require elongation of the target molecules (ligation step with an extension sequence), conversion of the target molecule into cDNA, as well as amplification steps. There are other approaches which are based on just a hybridisation step between targets and capture probes which tend to be long and increase the probability of obtaining false positives [10]. Therefore, the rapid and cost-effective detection and quantification of miRNAs, which will represent a huge advance and amelioration in diagnosis as it provides a high valuable knowledge to physicians, is still an unmet need. Real-time monitoring of miRNAs will become a key tool in personalised medicine [11].

An alternative approach for nucleic acid testing by dynamic chemistry (also known as Chem-NAT), which harnesses Watson-Crick base pairing to template a dynamic reaction on a strand of abasic peptide nucleic acid (PNA) probes [12] and using reactive nucleobases (SMART-NBs) has been developed by our group. During the past years, we have validated this approach to detect DNA mutations [13], [14], [15], [16] and miRNAs without the need of using PCR [17], [18], [19], [20]. Hence, new applications that fitted into the actual chemical biological and biomedicine demands are being developed. Herein, we aimed to develop a novel PCR-free method, based on Chem-NAT, for the rapid detection and quantification of miR-21 directly from tumour cell lines.

In our work, we have selected two widely and commonly used cancer cell lines, obtained by cell culturing, MDA-MB-468 (breast cancer mammalian cell line) and H1975 (lung cancer cell line), both overexpressing miR-21, and Peripheral Blood Mononuclear Cells (PBMCs) as control cell line due to its miR-21 lack of expression. For the purpose of quantification the levels of miRNA presented in those cells, magnetic microspheres (Dynabeads m270) were covalently bound to abasic PNA complementary to miR-21 to afford Magbeads-miR21, comprising both dynamic chemistry and bead-based reagents. This combination has been shown to be very effective and promising for miRNA detection from biological sources [19], [20]. To develop the protocol for miR-21 direct detection and quantification from cell lines, we firstly focused on the detection of miR-21 using fluorescence-activated cell sorting (FACS) as the reading platform. Afterwards, and once the protocol to use Chem-NAT with bead-based reagents to detect miR-21 was validated using FACS, we aimed to go one step further and quantify the number of miR-21 per cell. We decided to use a fluorescent microplate reader (FLUOstar Omega) rather than FACS to do miR-21 quantification as it offers higher throughput capacity.

This new application allows using cell pellets directly, avoiding miRNA isolation and purification steps, which reduces the possibility of contamination, and using fluorescent-based readout platforms (Fig. 1). This new proof-of-concept opens up the possibility for not only single-point miRNA quantification but for multiple points quantification, enabling miRNA level changes to be detected, and quantified, within cell-based assays. This approach, although it requires optimisation and further experiments, could represent a starting point for real monitoring of miRNA, what, as aforementioned, becomes a key tool in personalised medicine.

Section snippets

General

DGL probes were designed and synthesised by DestiNA Genomica S.L. (Spain), following standard solid phase chemistry protocols, using an INTAVIS MultiPrep Synthesizer (Intavis AG Gmb H, Germany). Mimic sequences of miRNA were purchased from Integrated DNA Technologies (IDT) as ssDNA (Table 1).

The microspheres Dynabeads® M-270 Carboxylic Acid were purchased from Thermofisher Scientific. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Sigma-Aldrich.

Flow Cytometry (FACS)

Cell lines were lysated and incubated with Magbeads-miR21 for miR-21 hybridisation (Hybridisation - Fig. 1). After pelleting out and washing Magbeads-miR21 from the cell lysate buffer, dynamic chemistry reaction was performed, using biotinylated aldehyde-modified SMART-NBs, which could be covalently attached to the abasic probe into the abasic position, through the templating role of complementary miRNA strand (DestiNA reaction – Fig. 1). Finally, bead labelling was performed using

Conclusions

We have presented a new Chem-NAT application which allows quantifying the number of miRNA per cell, without the need for miRNA isolation, following an easy-to-implement protocol in any cell biology lab. Concretely, we were able to detect and quantify the number of molecules of miR-21 per cell, a key miRNA involved in tumorigenic processes, in two tumour cancer cell lines, MDA-MB-468 and H1975, in less than 3 h and a half. Likewise, we have also confirmed that the expression of miR-21 in PBMCs

Acknowledgements

This research was supported by the Regional Government, Spain (BIO-1778) and the Ministry of Economy and Competitiveness, Spain (BIO2016-80519-R – FJLD is supported by Torres Quevedo fellowship, Spain PTQ-16-08597). ADG and ARR are supported by Spanish Ministry of Education, Spain (PhD scholarships FPU14/02181 and FPU15/06418, respectively). AMR thanks DestiNA Genomics, Spain and the University of Granada, Spain for a PhD scholarship (P26 Knowledge Transfer Programme) DMP acknowledges the

Contribution on each co-author

ADG: Designed, performed most of the experiments and wrote the paper

ARR: PCR experiments

AMR: Protocol optimisation with FLUOstar Omega

SD: Beads conjugation for FACS studies

FJLD: Synthesis of SMART-Nucleobases

DMP: PBMCs collection

JJGM: Probe synthesis and purification

MAF: Designed synthesis pathways for SMART Nucleobases and optimisation of protocols for abasic PNA synthesis

BLL & MTC: miR-21 probes validation and Dynamic incorporation optimisation

SP: Designed the experiments

RMSM: Designed the

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