DNA結合色素化学と液滴デジタルPCRを使用したマイクロRNA定量循環
Summary
A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.
Abstract
マイクロRNA（miRNAは）（無細胞の）循環血液流に細胞から放出されます。循環中の特定のマイクロRNAの量は、疾患状態に連結され、疾患のバイオマーカーとして使用する可能性を有するされています。デジタルPCR技術染料系化学を用いて、液滴マイクロRNA定量を循環させるための高感度で正確な方法が最近開発されました。具体的には、ロックされた核酸（LNA）を使用することは、特定のmiRNAの絶対的な定量化を得ることができる互換性のある液滴デジタルPCRシステムにおいて緑色蛍光DNA結合色素とのmiRNA特異的プライマーをベース。ここでは、そのような血漿および血清などの生物学的流体、中のmiRNAの量を評価するためにこの技術を実行方法について説明し、実行可能かつ効果的です。
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
循環するmiRNAは、非常に低濃度で血液中に存在し、血漿および血清試料から抽出することができるRNAの量は低いです。この理由のため、それらは、マイクロアレイおよびRNA配列のような他の技術を用いて定量化することは困難です。また、データの正規化と血液中の内因性の「基準」のmiRNAの存在に合意の一般化が欠如しています。この文脈において、液滴デジタルPCRのような敏感な技術は、…
Disclosures
The authors have nothing to disclose.
Acknowledgements
（ – 2010/15、9980ミルのn個当たり5。特別プログラム分子臨床腫瘍）と命令のイタリア省から、大学や癌研究のためのイタリア語協会（AIRC）からMF（MFAG 11676）にし、MNに資金を提供することによってサポートされていますMNへの研究FIRB 2011（プロジェクト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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