РНК-след Анализ Transcriptomes в тромбин обращению и контролю человека легочной микрососудистой эндотелиальных клеток
Summary
Этот протокол представляет собой полный и подробный порядок применения РНК-след, мощный следующего поколения секвенирования ДНК технологии, профиль transcriptomes в человеческой легочной микрососудистых эндотелиальных клеток с лечением или без тромбина. Этот протокол является обобщением различных клеток или тканей под влиянием различных реагентов или болезненных состояний.
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
Ключевые шаги
РНК-разгрузочные работы: РНКаз будет деградировать даже самого высокого качества РНК, поэтому необходимо соблюдать осторожность во время выделения, хранения и использования РНК-10. Перчатки всегда носили, чтобы предотвратить загрязнение РНК?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Авторы хотели бы поблагодарить д-р Стивен Kingsmore и детской Центра медицины генома в Детской милосердия Больницы и клиники для использования их вычислительных кластеров для анализа данных, поле Illumina служба команды (Elizabeth Бойер, Скотт Кук и Марк Кук) и технические Консультант команды для их быстрого реагирования и полезные советы на функционирование нового поколения секвенирования ДНК инструмент, HiScanSQ, а также данные анализа качества. Эта работа была выполнена при частичной поддержке Национального института здоровья Грант HL080042 (для SQY) и пуско-наладочные фонд и фонд детского милосердия Больницы и клиники Университета Миссури в Канзас-Сити (в SQY).
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. |
Riferimenti
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