Summary

थ्रोम्बिन इलाज में आरएनए - seq Transcriptomes का विश्लेषण और नियंत्रण मानव फेफड़े Microvascular endothelial कोशिकाओं

Published: February 13, 2013
doi:

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

इस प्रोटोकॉल की रूपरेखा प्रवाह संचित्र चित्र 1 में प्रदर्शित किया जाता है. 1. कोशिकाओं की थ्रोम्बिन, शाही सेना अलगाव, गुणवत्ता और शाही सेना का आकलन मात्रा के साथ उपचार संस्कृति मा?…

Representative Results

चरण 1: 28S: 18s अनुपात परंपरागत रूप से शाही सेना गिरावट का एक संकेतक के रूप में प्रयोग किया जाता है. आदर्श रूप में, 28S शिखर 18s बैंड (2 के एक अनुपात) के लगभग दो बार क्षेत्र में होना चाहिए, लेकिन यह आदर्श अनुपात ?…

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.

References

  1. Wang, Z., Gerstein, M., Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10, 57-63 (2009).
  2. Shendure, J., Ji, H. Next-generation DNA sequencing. Nat. Biotechnol. 26, 1135-1145 (2008).
  3. Marioni, J. C., Mason, C. E., Mane, S. M., Stephens, M., Gilad, Y. RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 18, 1509-1517 (2008).
  4. Zhang, L. Q., et al. RNA-seq Reveals Novel Transcriptome of Genes and Their Isoforms in Human Pulmonary Microvascular Endothelial Cells Treated with Thrombin. PLoS One. 7, e31229 (2012).
  5. Trapnell, C., Pachter, L., Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 25, 1105-1111 (2009).
  6. Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
  7. Li, H., et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 25, 2078-2079 (2009).
  8. Trapnell, C., et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511-515 (2010).
  9. Khatri, P., Sirota, M., Butte, A. J. Ten years of pathway analysis: current approaches and outstanding challenges. PLoS Comput. Biol. 8, e1002375 (2012).
  10. Nielsen, H. Working with RNA. Methods. Mol. Biol. 703, 15-28 (2011).
  11. Trapnell, C., et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562-578 (2012).
  12. Robertson, G., et al. De novo assembly and analysis of RNA-seq data. Nat. Methods. 7, 909-912 (2010).
  13. Garber, M., Grabherr, M. G., Guttman, M., Trapnell, C. Computational methods for transcriptome annotation and quantification using RNA-seq. Nat. Methods. 8, 469-477 (2011).
  14. Martin, J. A., Wang, Z. Next-generation transcriptome assembly. Nat. Rev. Genet. 12, 671-682 (2011).

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Cite This Article
Cheranova, D., Gibson, M., Chaudhary, S., Zhang, L. Q., Heruth, D. P., Grigoryev, D. N., Qing Ye, S. RNA-seq Analysis of Transcriptomes in Thrombin-treated and Control Human Pulmonary Microvascular Endothelial Cells. J. Vis. Exp. (72), e4393, doi:10.3791/4393 (2013).

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