Circulant microARN Quantification Utilisation DNA-binding Dye Chemistry and Droplet PCR numérique
Summary
A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.
Abstract
Circulant (des sans cellules) microARN (miARN) sont libérés à partir de cellules dans le flux sanguin. La quantité de micro-ARN spécifique dans la circulation a été liée à un état pathologique et a le potentiel pour être utilisé en tant que marqueur biologique de la maladie. Une méthode sensible et précis pour la circulation microRNA quantification en utilisant une chimie à base de colorants et des gouttelettes de la technologie PCR numérique a été récemment mis au point. Plus précisément, à l'aide Locked Nucleic Acid (LNA) à base d'amorces spécifiques de miARN avec un colorant fluorescent de liaison à l'ADN vert dans un système de PCR compatible avec des gouttelettes numérique, il est possible d'obtenir la quantification absolue des miARN spécifiques. Ici, nous décrivons comment effectuer cette technique pour évaluer le montant de miARN dans les liquides biologiques, tels que le plasma et le sérum, est à la fois faisable et efficace.
Introduction
MicroRNAs (miRNAs) are released into blood circulation by potentially all the cells of the organism, as a consequence of active release or necrotic and apoptotic processes. Cell-free miRNAs have been detected in the bloodstream either as free stable molecules or linked to lipoproteins or enveloped inside exosomes and microvesicles 1-3. They are believed to function as cell-to-cell communicators 4, and their amount changes in the presence of cancer, cardiac disorders or autoimmune diseases 5-7. Their accurate and reproducible quantification is the basis for their evaluation as disease biomarkers. However, for several reasons already described elsewhere 8,9, miRNA quantification in serum or plasma, as well as other body fluids, could be very challenging 10,11. We recently developed a method for the absolute quantification of circulating miRNAs, based on miRNA-specific LNA primers and DNA-binding dye droplet digital PCR (ddPCR) technology 12. This methodology has been applied to the validation of miRNA breast cancer biomarkers 13,14.
After the partitioning of each reverse-transcribed miRNA molecule inside a nanoliter-sized droplet, it is possible to count the copy number of each miRNA in each sample, basically counting the number of green, and therefore positive, fluorescent droplets. As soon as a PCR reaction occurs, a positive count is achieved, without the need to establish a standard curve or taking PCR efficiency into account in target amount calculation, as it happens with quantitative RT-PCR (RT-qPCR). In addition, ddPCR proved to be more sensitive and accurate than RT-qPCR in circulating miRNA quantification 15. In this article we present the detailed protocol of this methodology, discussing the most relevant steps andspecifically considering serum and plasma clinical samples.
Protocol
Representative Results
Discussion
miARN circulantes sont présents dans le sang à des concentrations extrêmement faibles et la quantité d'ARN qui peut être extrait à partir d'échantillons de plasma et de sérum est faible. Pour cette raison, il est difficile de quantifier avec d'autres techniques telles que la puce à ADN et le séquençage de l'ARN. De plus, il y a un manque généralisé d'accord sur la normalisation des données et la présence de miARN "de référence" endogènes dans le sang. Dans ce contexte, un…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Soutenu par un financement de l'Association italienne pour la recherche sur le cancer (AIRC) MF (MFAG 11676) et MN (Molecular Programme spécial Clinical Oncology -. 5 pour mille n 9980, 2010/15) et du ministère italien de l'Instruction, de l'Université et Research FIRB 2011 MN (projet RBAPIIBYNP).
Materials
| miRNeasy Mini Kit | Qiagen | 217004 | Columns for total RNA, including miRNA, extraction from serum/plasma |
| 100 nmole RNA oligo Cel-miR-39-3p | Integrated DNA Technologies | Custom | Sequence: UCACCGGGUGUAAAUCAGCUUG |
| Universal cDNA synthesis kit II, 8-64 rxns | Exiqon | 203301 | Kit for microRNA reverse transcription |
| MicroRNA LNA PCR primer set | Exiqon | 204000-206xxx and 2100000-21xxxxx | Primers for miRNA amplification inside droplets |
| QX200 droplet generator | BioRad | 186-4002 | Instrument used for droplet reading |
| QX200 droplet reader | BioRad | 186-4003 | Instrument used for droplet generation |
| QuantaSoft software | BioRad | 186-3007 | Software for data collection and analysis |
| PX1 PCR plate sealer | BioRad | 181-4000 | Plate sealer |
| DG8 droplet generator cartridges and gaskets | BioRad | 186-4008 | Cartridges used to mix sample and oil to generate droplets |
| QX200 ddPCR EvaGreen supermix | BioRad | 186-4033/36 | PCR supermix |
| QX200 droplet generator oil for EvaGreen dye | BioRad | 186-4005 | Oil for droplet generation |
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