RNA-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells

Dilyara Cheranova, Margaret Gibson, Suman Chaudhary, Li Qin Zhang, Daniel P. Heruth, Dmitry N. Grigoryev, Shui Qing Ye

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
JournalJournal of Visualized Experiments
Issue number72
DOIs
StatePublished - Feb 13 2013
Externally publishedYes

Fingerprint

Endothelial cells
Gene Expression Profiling
RNA
Thrombin
Transcriptome
Endothelial Cells
Lung
Tissue
DNA
RNA Sequence Analysis
Genes
Drug therapy
Oligonucleotide Array Sequence Analysis
DNA Sequence Analysis
Libraries
Microarrays
Coronary Disease
Medical problems
Gene expression
Protein Isoforms

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Cheranova, D., Gibson, M., Chaudhary, S., Zhang, L. Q., Heruth, D. P., Grigoryev, D. N., & Ye, S. Q. (2013). RNA-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells. Journal of Visualized Experiments, (72). https://doi.org/10.3791/4393

RNA-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells. / Cheranova, Dilyara; Gibson, Margaret; Chaudhary, Suman; Zhang, Li Qin; Heruth, Daniel P.; Grigoryev, Dmitry N.; Ye, Shui Qing.

In: Journal of Visualized Experiments, No. 72, 13.02.2013.

Research output: Contribution to journalArticle

Cheranova, Dilyara ; Gibson, Margaret ; Chaudhary, Suman ; Zhang, Li Qin ; Heruth, Daniel P. ; Grigoryev, Dmitry N. ; Ye, Shui Qing. / RNA-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells. In: Journal of Visualized Experiments. 2013 ; No. 72.
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