मानक प्रयोगशाला रिवर्स ट्रांसक्रिपटेस प्रतिक्रियाओं में mRNA की एकल कोशिका मात्रा से सीडीएनए पैदावार बढ़ाने ध्वनिक Microstreaming का उपयोग
Summary
हम mRNA की एकल कोशिका मात्रा से अन्यथा मानक प्रयोगशाला रिवर्स प्रतिलेखन प्रतिक्रियाओं में सीडीएनए उपज बढ़ाने के लिए एक उपन्यास विधि का वर्णन. नवीनता micromixer, जो ध्वनिक microstreaming की घटना का इस्तेमाल का उपयोग में रहता है, मिलाते हुए, vortexing या trituration से microliter पैमाने पर तरल पदार्थ और अधिक प्रभावी ढंग से मिश्रण.
Abstract
Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50pg total RNA, or 0.01-2.5pg mRNA, per cell 1) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell 2. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used 3,4. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields 5-8. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel “micromixing” technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: (1) greater numbers of genes to be analyzed per cell; (2) more quantitative analysis of gene expression; and (3) better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming 9-12, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device (“micromixer”) with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature 13. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile 13, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.
Protocol
Discussion
मानक प्रयोगशाला आरटी प्रतिक्रियाओं micromixing के आवेदन की विधि यहाँ वर्णित है, ज़ाहिर है, को शामिल कर सकते हैं किसी भी विधि (जैसे सेल lysis, लेजर कब्जा microdissection) के माध्यम से काटा mRNA. यह भी किसी भी ब्रांड या आरटी अभिकर्…
Disclosures
The authors have nothing to disclose.
Acknowledgements
यह अध्ययन राष्ट्रीय स्वास्थ्य और ऑस्ट्रेलिया के चिकित्सा अनुसंधान परिषद (परियोजना को कोई अनुदान 6,288,480) और Scobie और क्लेयर MacKinnon ट्रस्ट द्वारा समर्थित किया गया था.
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| Total RNA was isolated from snap frozen acutely prepared adult mouse midbrain slices | |||
| PicoPure RNA Isolation Kit | Arcturus, CA, USA | KIT0204 | The kit is now available from Applied Biosystems |
| DNA-free DNase Treatment and Removal Reagents | Ambion | AM1906M | |
| Random hexamer primers | Promega | C1181 | |
| AMV-RT | Promega | M5101 | |
| dNTP set | Promega | U1240 | |
| RNasin Ribonuclease Inhibitor | Promega | N2111 | |
| Nuclease-Free Water | Promega | P1193 | |
| SYBR Green PCR Master Mix | Applied Biosystem | 4309155 | |
| Hprt forward (20mer):CTT TGC TGA CCT GCT GGA TT | |||
| Hprt reverse (20mer):TAT GTC CCC CGT TGA CTG AT | |||
| Nurr1 forward (23mer):CAG CTC CGA TTT CTT AAC TCC AG | |||
| Nurr1 reverse (23mer):GGT GAG GTC CAT GCT AAA CTT GA | |||
| NanoDrop 1000 Spectrophotometer. | Thermo Scientific | ||
| Corbett Rotor Gene RG-6000 | Corbett Life Science | Corbett Life Science was acquired by QIAGEN in July 2008 |
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