थ्रोम्बिन इलाज में आरएनए - seq Transcriptomes का विश्लेषण और नियंत्रण मानव फेफड़े Microvascular endothelial कोशिकाओं
Summary
यह प्रोटोकॉल का एक पूर्ण और विस्तृत प्रक्रिया मानव थ्रोम्बिन उपचार के साथ या बिना फेफड़े microvascular endothelial कोशिकाओं में प्रोफाइल transcriptomes आरएनए seq, एक शक्तिशाली अगली पीढ़ी के डीएनए अनुक्रमण प्रौद्योगिकी, लागू प्रस्तुत करता है. यह प्रोटोकॉल विभिन्न कोशिकाओं या ऊतकों अलग अभिकर्मकों या रोग राज्यों द्वारा प्रभावित generalizable है.
Abstract
The characterization of gene expression in cells via measurement of mRNA levels is a useful tool in determining how the transcriptional machinery of the cell is affected by external signals (e.g. drug treatment), or how cells differ between a healthy state and a diseased state. With the advent and continuous refinement of next-generation DNA sequencing technology, RNA-sequencing (RNA-seq) has become an increasingly popular method of transcriptome analysis to catalog all species of transcripts, to determine the transcriptional structure of all expressed genes and to quantify the changing expression levels of the total set of transcripts in a given cell, tissue or organism1,2 . RNA-seq is gradually replacing DNA microarrays as a preferred method for transcriptome analysis because it has the advantages of profiling a complete transcriptome, providing a digital type datum (copy number of any transcript) and not relying on any known genomic sequence3.
Here, we present a complete and detailed protocol to apply RNA-seq to profile transcriptomes in human pulmonary microvascular endothelial cells with or without thrombin treatment. This protocol is based on our recent published study entitled “RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin,”4 in which we successfully performed the first complete transcriptome analysis of human pulmonary microvascular endothelial cells treated with thrombin using RNA-seq. It yielded unprecedented resources for further experimentation to gain insights into molecular mechanisms underlying thrombin-mediated endothelial dysfunction in the pathogenesis of inflammatory conditions, cancer, diabetes, and coronary heart disease, and provides potential new leads for therapeutic targets to those diseases.
The descriptive text of this protocol is divided into four parts. The first part describes the treatment of human pulmonary microvascular endothelial cells with thrombin and RNA isolation, quality analysis and quantification. The second part describes library construction and sequencing. The third part describes the data analysis. The fourth part describes an RT-PCR validation assay. Representative results of several key steps are displayed. Useful tips or precautions to boost success in key steps are provided in the Discussion section. Although this protocol uses human pulmonary microvascular endothelial cells treated with thrombin, it can be generalized to profile transcriptomes in both mammalian and non-mammalian cells and in tissues treated with different stimuli or inhibitors, or to compare transcriptomes in cells or tissues between a healthy state and a disease state.
Protocol
Representative Results
Discussion
प्रमुख कदम
आरएनए हैंडलिंग: RNases भी सबसे उच्च गुणवत्ता वाले शाही सेना नीचा, इसलिए देखभाल अलगाव, भंडारण और 10 शाही सेना के उपयोग के दौरान लिया जाना चाहिए. दस्ताने को हमेशा के लिए मानव हाथ ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए बच्चों की दया अस्पतालों और क्लीनिकों में डॉ. स्टीफन Kingsmore और बाल जीनोम चिकित्सा केंद्र हमारे डेटा विश्लेषण के लिए अपने कंप्यूटिंग समूहों के उपयोग के लिए शुक्रिया अदा करना चाहूँगा, Illumina सेवा क्षेत्र (एलिजाबेथ बोयर, स्कॉट कुक और मार्क कुक) टीम और तकनीकी उनके त्वरित प्रतिक्रिया और अगली पीढ़ी के डीएनए अनुक्रमण साधन, HiScanSQ, और डेटा की गुणवत्ता विश्लेषण के चलने पर उपयोगी सुझाव के लिए सलाहकार टीम. इस काम के हिस्से में स्वास्थ्य HL080042 ग्रांट (SQY करने के लिए) के राष्ट्रीय संस्थानों द्वारा समर्थित किया गया था और शुरू हुआ और बच्चों की दया अस्पतालों और क्लीनिकों की निधि बंदोबस्ती, कैनसस सिटी में मिसौरी विश्वविद्यालय (SQY करने के लिए).
Materials
| Reagents or Equipments | Company | Catalog number | Comments |
| Human Lung Microvascular Endothelial Cells | Lonza | CC-2815 | |
| Lonza, Bullet Kit | Lonza | CC-3202 | Contains EGM-2, FBS, growth factors and antibiotics |
| Thrombin | Sigma | T4393 | |
| Ambion mirVana Kit | Life Technologies | AM 1560 | |
| RNase-Zap | Life Technologies | AM9782 | |
| Experion StdSens RNA | Bio-Rad | 700-7103 | |
| TruSeq RNA Preparation Kit | Illumina | FC-122-1001 | |
| AMPureXP Beads | Beckman Coulter | A63881 | |
| Superscript Reverse Transcriptase II | Life Technologies | 18064-014 | |
| Experion DNA 1K | Bio-Rad | 700-7107 | |
| QuantiTect SyberGreen | Qiagen | 204163 | |
| PE Cluster Generation Kit | Illumina | PE-401-3001 | |
| PhiX Control Kit | Illumina | FC110-301 | |
| 200 Cycle SBS Kit | Illumina | FC-401-3001 | |
| HiScanSQ* | Illumina | SY-103-2001 | |
| cBot | Illumina | SY-301-2002 | |
| qPCR machine – Viia7 | Life Technologies | Model #VIIA7 / Equipment #10631261 | Or equivalent |
| Experion System | Bio Rad | 7007001 | Bioanalyzer is an alternative system |
| Spectrophotometer | Bio-Tek | Epoch Microplate Spectrophotometer | Or equivalent |
| Centrifuge – Sorvall Legend XTR | Thermo Scientific | 75004521 | Or equivalent |
| Magnetic stand | Life Technologies | AM10027 | |
| 96-well thermocycler | General Lab Supplier | ||
| Table 3. List of Key Reagents and Major Equipment. *, In the video, HiSeq1000 instead of HiScanSQ was demonstrated. |
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