Transcriptome profiling of refractory atopic keratoconjunctivitis by RNA sequencing

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

Fusion transcriptome profiling provides insights into alveolar rhabdomyosarcoma

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1612734113 ◽

2016 ◽

Vol 113 (46) ◽

pp. 13126-13131 ◽

Cited By ~ 21

Author(s):

Zhongqiu Xie ◽

Mihaela Babiceanu ◽

Shailesh Kumar ◽

Yuemeng Jia ◽

Fujun Qin ◽

...

Keyword(s):

Rna Sequencing ◽

Fusion Gene ◽

Transcriptome Profiling ◽

Alveolar Rhabdomyosarcoma ◽

Clinical Samples ◽

Cell Of Origin ◽

Rna Profiling ◽

Chimeric Rnas ◽

Fusion Products ◽

Time Point

Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3–FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3–FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3–FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3–FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.

Download Full-text

Elucidating the microRNA-203 specific biological processes in glioblastoma cells from comprehensive RNA-sequencing transcriptome profiling

Cellular Signalling ◽

10.1016/j.cellsig.2018.09.014 ◽

2019 ◽

Vol 53 ◽

pp. 22-38 ◽

Cited By ~ 2

Author(s):

Bhavesh K. Ahir ◽

Sajani S. Lakka

Keyword(s):

Rna Sequencing ◽

Transcriptome Profiling ◽

Biological Processes ◽

Glioblastoma Cells

Download Full-text

Integrative Transcriptome Profiling Reveals SKA3 as a Novel Prognostic Marker in Non-Muscle Invasive Bladder Cancer

Cancers ◽

10.3390/cancers13184673 ◽

2021 ◽

Vol 13 (18) ◽

pp. 4673

Author(s):

Chaelin You ◽

Xuan-Mei Piao ◽

Keunsoo Kang ◽

Yong-June Kim ◽

Kyuho Kang

Keyword(s):

Bladder Cancer ◽

Cancer Patients ◽

Rna Sequencing ◽

Clinical Outcomes ◽

Prognostic Marker ◽

Transcriptome Profiling ◽

Invasive Bladder Cancer ◽

Cell Subpopulation ◽

Muscle Invasive Bladder Cancer ◽

Muscle Invasive

Approximately 80% of all new bladder cancer patients are diagnosed with non-muscle invasive bladder cancer (NMIBC). However, approximately 15% of them progress to muscle-invasive bladder cancer (MIBC), for which prognosis is poor. The current study aimed to improve diagnostic accuracy associated with clinical outcomes in NMIBC patients. Nevertheless, it has been challenging to identify molecular biomarkers that accurately predict MIBC progression because this disease is complex and heterogeneous. Through integrative transcriptome profiling, we showed that high SKA3 expression is associated with poor clinical outcomes and MIBC progression. We performed RNA sequencing on human tumor tissues to identify candidate biomarkers in NMIBC. We then selected genes with prognostic significance by analyzing public datasets from multiple cohorts of bladder cancer patients. We found that SKA3 was associated with NMIBC pathophysiology and poor survival. We analyzed public single-cell RNA-sequencing (scRNA-seq) data for bladder cancer to dissect transcriptional tumor heterogeneity. SKA3 was expressed in an epithelial cell subpopulation expressing genes regulating the cell cycle. Knockdown experiments confirmed that SKA3 promotes bladder cancer cell proliferation by accelerating G2/M transition. Hence, SKA3 is a new prognostic marker for predicting NMIBC progression. Its inhibition could form part of a novel treatment lowering the probability of bladder cancer progression.

Download Full-text