RESUMEN
A novel sensitive, specific and rapid method for the detection and quantification of microRNAs without requiring extraction from their biological sources is now available using a novel chemical based, PCR-free technology for nucleic acid testing. In this study, we both demonstrate how this method can be used to profile miR-451a, an important miRNA in erythropoiesis, and compare with the gold standard RT-qPCR.
Asunto(s)
Biomarcadores de Tumor/sangre , MicroARNs/sangre , Hibridación de Ácido Nucleico/métodos , Espectrometría de Fluorescencia/métodos , Biomarcadores de Tumor/genética , Galactósidos/química , Humanos , Límite de Detección , MicroARNs/genética , Oxazinas/química , beta-Galactosidasa/químicaRESUMEN
The detection of repetitive sequences with single-base resolution is becoming increasingly important aiming to understand the biological implications of genomic variation in these sequences. However, there is a lack of techniques to experimentally validate sequencing data from repetitive sequences obtained by Next-Generation Sequencing methods, especially in the case of Single-Nucleotide Variations (SNVs). That is one of the reasons why repetitive sequences have been poorly studied and excluded from most genomic studies. Therefore, in addition to sequencing data, there is an urgent need for efficient validation methods of genomic variation in these sequences. Herein we report the development of chemFISH, an alternative method for the detection of SNVs in repetitive sequences. ChemFISH is an innovative method based on dynamic chemistry labelling and abasic Peptide Nucleic Acid (PNA) probes to detect in situ the α-satellite DNA, organized in tandem repeats, with single-base resolution in a direct and rapid reaction. With this approach, we detected by microscopy the α-satellite DNA in a variety of human cell lines, we quantified the detection showing a low coefficient of variation among samples (13.16%-25.33%) and we detected single-base specificity with high sensitivity (82.41%-88.82%). These results indicate that chemFISH can serve as a rapid method to validate previously detected SNVs in sequencing data, as well as to find novel SNVs in repetitive sequences. Furthermore, the versatile chemistry behind chemFISH can lead to develop novel molecular assays for the in situ detection of nucleic acids.
RESUMEN
Nucleic acid-based molecular diagnosis has gained special importance for the detection and early diagnosis of genetic diseases as well as for the control of infectious disease outbreaks. The development of systems that allow for the detection and analysis of nucleic acids in a low-cost and easy-to-use way is of great importance. In this context, we present a combination of a nanotechnology-based approach with the already validated dynamic chemical labeling (DCL) technology, capable of reading nucleic acids with single-base resolution. This system allows for the detection of biotinylated molecular products followed by simple detection using a standard flow cytometer, a widely used platform in clinical and molecular laboratories, and therefore, is easy to implement. This proof-of-concept assay has been developed to detect mutations in KRAS codon 12, as these mutations are highly important in cancer development and cancer treatments.
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Ácidos Nucleicos , Ácidos Nucleicos de Péptidos , Citometría de Flujo , Mutación , Nanotecnología , Ácidos Nucleicos/genéticaRESUMEN
Dynamic chemical labelling is a single-base specific method to enable detection and quantification of micro-Ribonucleic Acids in biological fluids without extraction and pre-amplification. In this study, dynamic chemical labelling was combined with the Luminex MAGPIX system to profile levels of microRNA-122 biomarker in serum from patients with Drug-Induced Liver Injury.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , MicroARNs , Biomarcadores , HumanosRESUMEN
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.