Di circolazione MicroRNA Quantificazione Utilizzando DNA-binding Dye Chimica e di gocce Digital PCR
Summary
A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.
Abstract
Di circolazione (di cell-free) microRNA (miRNA) vengono rilasciati dalle cellule nel flusso sanguigno. La quantità di microRNA specifici in circolazione è stato collegato a uno stato di malattia e ha il potenziale per essere utilizzato come biomarker della malattia. Un metodo sensibile e preciso per circolazione microRNA quantificazione utilizzando una chimica a base di colorante e di gocce tecnologia PCR digitale è stato recentemente sviluppato. In particolare, utilizzando Locked Nucleic Acid (LNA) a base di primer specifici miRNA con un colorante legante il DNA fluorescente verde in un sistema PCR compatibile gocciolina digitale, è possibile avere la quantificazione assoluta di miRNA specifici. Qui, descriviamo come l'esecuzione di questa tecnica per valutare la quantità di miRNA nei fluidi biologici, come il plasma e il siero, è sia fattibile ed 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
miRNA circolanti sono presenti nel sangue a concentrazioni estremamente basse e la quantità di RNA che può essere estratto da campioni di plasma e siero è basso. Per questo motivo, sono difficili da quantificare con altre tecniche come microarray e RNA sequencing. Inoltre, vi è una generalizzata mancanza di accordo sulla normalizzazione dei dati e la presenza di endogene miRNA "di riferimento" nel sangue. In questo contesto, una tecnologia sensibile come gocciolina PCR digitale, in grado di contare il nume…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Sostenuto da un finanziamento dell'Associazione Italiana per la Ricerca sul Cancro (AIRC) a MF (MFAG 11676) e di MN (Programma Speciale Oncologia Molecolare Clinica -. 5 per mille 9980 n, 2010/15) e dal Ministero Italiano dell'Istruzione, dell'Università e della La ricerca FIRB 2011 al MN (Progetto 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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