Understanding Disease Mechanisms of Hereditary Angioedema With Potential Therapeutic Implications Through RNA Sequencing Transcriptome Profiling

Abstract This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

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Targeted DNA and RNA Sequencing of Paired Urothelial and Squamous Bladder Cancers Reveals Discordant Genomic and Transcriptomic Events and Unique Therapeutic Implications

European Urology ◽

10.1016/j.eururo.2018.06.047 ◽

2018 ◽

Vol 74 (6) ◽

pp. 741-753 ◽

Cited By ~ 32

Author(s):

Daniel H. Hovelson ◽

Aaron M. Udager ◽

Andrew S. McDaniel ◽

Petros Grivas ◽

Phillip Palmbos ◽

...

Keyword(s):

Rna Sequencing ◽

Therapeutic Implications ◽

Dna And Rna

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Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1901572116 ◽

2019 ◽

Vol 116 (22) ◽

pp. 10824-10833 ◽

Cited By ~ 32

Author(s):

Sangbae Kim ◽

Albert Lowe ◽

Rachayata Dharmat ◽

Seunghoon Lee ◽

Leah A. Owen ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Cell Fate ◽

Time Course ◽

Transcriptome Profiling ◽

Retinal Cell ◽

Peripheral Retina ◽

Functional Validation ◽

Retinal Progenitor ◽

Retinal Differentiation

Rod and cone photoreceptors are light-sensing cells in the human retina. Rods are dominant in the peripheral retina, whereas cones are enriched in the macula, which is responsible for central vision and visual acuity. Macular degenerations affect vision the most and are currently incurable. Here we report the generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids differentiated from hESCs using an improved retinal differentiation system. Induced by extracellular matrix, aggregates of hESCs formed single-lumen cysts composed of epithelial cells with anterior neuroectodermal/ectodermal fates, including retinal cell fate. Then, the cysts were en bloc-passaged, attached to culture surface, and grew, forming colonies in which retinal progenitor cell patches were found. Following gentle cell detachment, retinal progenitor cells self-assembled into retinal epithelium—retinal organoid—that differentiated into stratified cone-rich retinal tissue in agitated cultures. Electron microscopy revealed differentiating outer segments of photoreceptor cells. Bulk RNA-sequencing profiling of time-course retinal organoids demonstrated that retinal differentiation in vitro recapitulated in vivo retinogenesis in temporal expression of cell differentiation markers and retinal disease genes, as well as in mRNA alternative splicing. Single-cell RNA-sequencing profiling of 8-mo retinal organoids identified cone and rod cell clusters and confirmed the cone enrichment initially revealed by quantitative microscopy. Notably, cones from retinal organoids and human macula had similar single-cell transcriptomes, and so did rods. Cones in retinal organoids exhibited electrophysiological functions. Collectively, we have established cone-rich retinal organoids and a reference of transcriptomes that are valuable resources for retinal studies.

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Transcriptome profiling of refractory atopic keratoconjunctivitis by RNA sequencing

Journal of Allergy and Clinical Immunology ◽

10.1016/j.jaci.2018.11.007 ◽

2019 ◽

Vol 143 (4) ◽

pp. 1610-1614.e6 ◽

Cited By ~ 4

Author(s):

Akira Matsuda ◽

Yosuke Asada ◽

Naomasa Suita ◽

Satoshi Iwamoto ◽

Toshiaki Hirakata ◽

...

Keyword(s):

Rna Sequencing ◽

Transcriptome Profiling ◽

Atopic Keratoconjunctivitis

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Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

Nature Communications ◽

10.1038/s41467-020-20448-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dandan Liang ◽

Jinfeng Xue ◽

Li Geng ◽

Liping Zhou ◽

Bo Lv ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Sinoatrial Node ◽

Induced Pluripotent Stem Cell ◽

Transcriptome Profiling ◽

Core Gene ◽

Cell Cluster ◽

Critical Function ◽

Gene Regulation Network ◽

Single Cell Rna Sequencing

AbstractBioelectrical impulses intrinsically generated within the sinoatrial node (SAN) trigger the contraction of the heart in mammals. Though discovered over a century ago, the molecular and cellular features of the SAN that underpin its critical function in the heart are uncharted territory. Here, we identify four distinct transcriptional clusters by single-cell RNA sequencing in the mouse SAN. Functional analysis of differentially expressed genes identifies a core cell cluster enriched in the electrogenic genes. The similar cellular features are also observed in the SAN from both rabbit and cynomolgus monkey. Notably, Vsnl1, a core cell cluster marker in mouse, is abundantly expressed in SAN, but is barely detectable in atrium or ventricle, suggesting that Vsnl1 is a potential SAN marker. Importantly, deficiency of Vsnl1 not only reduces the beating rate of human induced pluripotent stem cell - derived cardiomyocytes (hiPSC-CMs) but also the heart rate of mice. Furthermore, weighted gene co-expression network analysis (WGCNA) unveiled the core gene regulation network governing the function of the SAN in mice. Overall, these findings reveal the whole transcriptome profiling of the SAN at single-cell resolution, representing an advance toward understanding of both the biology and the pathology of SAN.

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