تعميم الرنا الميكروي الكمي باستخدام الحمض النووي ملزم صبغ الكيمياء والقطرة PCR الرقمية
Summary
A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.
Abstract
تعميم (من خالية من الخلايا) الرنا الميكروية (miRNAs) تم إصدارها من الخلايا في مجرى الدم. وقد تم ربط كمية من microRNAs محددة في الدورة الدموية إلى حالة المرض ولديه القدرة على أن تستخدم العلامات البيولوجية المرض. تم تطوير طريقة حساسة ودقيقة لتعميم الرنا الميكروي الكمي باستخدام الكيمياء قائم على الأصباغ وقطيرة تكنولوجيا PCR الرقمية مؤخرا. على وجه التحديد، وذلك باستخدام مغلق الأحماض النووية (LNA) المستندة الاشعال ميرنا معين مع الفلورسنت صبغة الحمض النووي ملزم الأخضر في قطرات نظام PCR الرقمية متوافق أنه من الممكن الحصول على تقدير المطلق للmiRNAs محددة. هنا، نحن تصف كيفية أداء هذه التقنية لتقييم كمية ميرنا في السوائل البيولوجية، مثل البلازما والمصل، على حد سواء مجدية وفعالة.
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
miRNAs تعميم موجودة في الدم بتركيزات منخفضة للغاية وكمية من الحمض النووي الريبي التي يمكن استخلاصها من عينات البلازما والمصل منخفضة. لهذا السبب، فإن من الصعب تحديد مع تقنيات أخرى مثل ميكروأري وتسلسل الحمض النووي الريبي. وعلاوة على ذلك، هناك نقص معمم الاتفاق على تطبيع ا…
Disclosures
The authors have nothing to disclose.
Acknowledgements
بدعم من تمويل من الاتحاد الإيطالي لأبحاث السرطان (جنة العلاقات الصناعية الاسترالية) لMF (MFAG 11676) وMN (برنامج الجزيئية الخاصة الأورام السريري – 5 بالألف ن 9980، 2010/15) ومن الوزارة الإيطالية للتعليم والجامعات و البحث FIRB 2011 إلى MN (مشروع 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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