परिसंचारी MicroRNA मात्रा डीएनए बाध्यकारी डाई रसायन विज्ञान और छोटी बूंद डिजिटल पीसीआर का उपयोग
Summary
A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.
Abstract
microRNAs (miRNAs) (सेल मुक्त का) परिसंचारी रक्त प्रवाह में कोशिकाओं से जारी कर रहे हैं। संचलन में विशिष्ट microRNAs की राशि एक रोग राज्य से जोड़ा गया है और संभावित रोग बायोमार्कर के रूप में इस्तेमाल किया जाना है। microRNA मात्रा का ठहराव घूम एक डाई आधारित रसायन विज्ञान का उपयोग और डिजिटल पीसीआर प्रौद्योगिकी छोटी बूंद के लिए एक संवेदनशील और सटीक विधि हाल ही में विकसित किया गया है। विशेष रूप से, बंद का उपयोग न्यूक्लिक एसिड (LNA) एक संगत छोटी बूंद डिजिटल पीसीआर प्रणाली यह विशिष्ट miRNAs की पूर्ण मात्रा का ठहराव प्राप्त करने के लिए संभव है में एक हरी फ्लोरोसेंट डीएनए बाध्यकारी डाई के साथ miRNA विशिष्ट प्राइमरों आधारित। यहाँ, हम वर्णन कैसे इस तकनीक का प्रदर्शन ऐसे प्लाज्मा और सीरम के रूप में जैविक तरल पदार्थ, में miRNA राशि का आकलन करने के लिए, दोनों व्यावहारिक और प्रभावी है।
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
परिसंचारी miRNAs बहुत कम मात्रा में रक्त में मौजूद हैं और शाही सेना की राशि है कि प्लाज्मा और सीरम नमूनों से निकाला जा सकता है कम है। इस कारण से, वे इस तरह के माइक्रोएरे और आरएनए अनुक्रमण के रूप में अन्य तकनी?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
इतालवी (AIRC) म्यूचुअल फंड (MFAG 11676) के कैंसर रिसर्च के लिए एसोसिएशन से और एम.एन. को धन के द्वारा समर्थित (विशेष कार्यक्रम आण्विक नैदानिक कैंसर विज्ञान -। प्रति सहस्र N 5 9980, 2010/15) और निर्देश के इतालवी मंत्रालय, विश्वविद्यालय और से अनुसंधान FIRB 2011 एम.एन. करने के लिए (परियोजना 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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