scholarly journals A method for simultaneous detection of small and long RNA biotypes by ribodepleted RNA-Seq

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Nikita Potemkin ◽  
Sophie M. F. Cawood ◽  
Jackson Treece ◽  
Diane Guévremont ◽  
Christy J. Rand ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Protein Coding ◽  
Biologically Relevant ◽  
High Loss ◽  
Non Coding Rna ◽  
Tissue Removal ◽  
Rrna Depletion ◽  
High Integrity

AbstractRNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was extremely efficient, resulting in less than 1.5% rRNA content in the final library. We identified > 30,000 unique transcripts from all samples, including protein-coding genes and many species of non-coding RNA, in biologically-relevant proportions. Furthermore, the normalized sequencing read count for select genes significantly negatively correlated with Ct values from qRT-PCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

scholarly journals A workflow for simultaneous detection of coding and non-coding transcripts by ribosomal RNA-depleted RNA-Seq.

2021 ◽  
Author(s):  
Joanna M. Williams ◽  
Nikita Potemkin ◽  
Sophie Cawood ◽  
Jackson Treece ◽  
Diane Guévremont ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Rna Seq ◽  
Protein Coding ◽  
Biologically Relevant ◽  
High Loss ◽  
Highly Efficient ◽  
Non Coding Rna ◽  
Tissue Removal ◽  
Rrna Depletion ◽  
Unique Species

RNA sequencing offers unprecedented access to the transcriptome. Key to this is the identification and quantification of many different species of RNA from the same sample at the same time. In this study we describe a novel protocol for simultaneous detection of coding and non-coding transcripts using modifications to the Ion Total RNA-Seq kit v2 protocol, with integration of QIASeq FastSelect rRNA removal kit. We report highly consistent sequencing libraries can be produced from both frozen high integrity mouse hippocampal tissue and the more challenging post-mortem human tissue. Removal of rRNA using FastSelect was highly efficient, resulting in less than 1.5% rRNA content in the final library, significantly better than other reported rRNA removal techniques. We identified >30,000 unique transcripts from all samples, including protein-coding genes and many unique species of non-coding RNA, in biologically-relevant proportions. Furthermore, normalized sequencing read count for select genes significantly negatively correlated with Ct values from RT-qPCR analysis from the same samples. These results indicate that this protocol accurately and consistently identifies and quantifies a wide variety of transcripts simultaneously. The highly efficient rRNA depletion, coupled with minimized sample handling and without complicated and high-loss size selection protocols, makes this protocol useful to researchers wishing to investigate whole transcriptomes.

scholarly journals Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries

2021 ◽  
Author(s):  
Calla L Telzrow ◽  
Paul J Zwack ◽  
Shannon Esher Righi ◽  
Fred S Dietrich ◽  
Cliburn Chan ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Rna Sequencing ◽  
Rnase H ◽  
Rrna Genes ◽  
Rna Seq ◽  
Protein Coding ◽  
Mitochondrial Rrna ◽  
Non Coding Rna ◽  
Rrna Depletion ◽  
Enrichment Methods

Abstract RNA sequencing (RNA-Seq) experiments focused on gene expression involve removal of ribosomal RNA (rRNA) because it is the major RNA constituent of cells. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing must be performed to compensate for the sequencing reads wasted on rRNA. The ideal RNA enrichment method removes all rRNA without affecting other RNA in the sample. We tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We find that the RNase H depletion method is more efficient in depleting rRNA and more specific in recapitulating non-rRNA levels present in unenriched controls than the commonly-used Poly(A) isolation method. The RNase H depletion method is also more effective than the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding RNA levels present in unenriched controls, while the Ribo-Zero depletion method more closely recapitulates annotated non-coding RNA (ncRNA) levels. Finally, we leverage these data to accurately map the C. neoformans mitochondrial rRNA genes, and also demonstrate that RNA-Seq data generated with the RNase H and Ribo-Zero depletion methods can be used to explore novel C. neoformans long non-coding RNA genes.

scholarly journals Non-Coding RNA Signatures of B-Cell Acute Lymphoblastic Leukemia

2021 ◽  
Vol 22 (5) ◽  
pp. 2683
Author(s):  
Princess D. Rodriguez ◽  
Hana Paculova ◽  
Sophie Kogut ◽  
Jessica Heath ◽  
Hilde Schjerven ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Lymphoblastic Leukemia ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Technological Developments ◽  
Cell Acute Lymphoblastic Leukemia

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell acute lymphoblastic leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

scholarly journals Emerging Roles of Estrogen-Regulated Enhancer and Long Non-Coding RNAs

2020 ◽  
Vol 21 (10) ◽  
pp. 3711
Author(s):  
Melina J. Sedano ◽  
Alana L. Harrison ◽  
Mina Zilaie ◽  
Chandrima Das ◽  
Ramesh Choudhari ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Expression Patterns ◽  
Biological Significance ◽  
Rna Seq ◽  
Biological Functions ◽  
Protein Coding ◽  
Rna Molecules ◽  
Non Coding Rna ◽  
Genome Wide ◽  
Non Coding Rnas

Genome-wide RNA sequencing has shown that only a small fraction of the human genome is transcribed into protein-coding mRNAs. While once thought to be “junk” DNA, recent findings indicate that the rest of the genome encodes many types of non-coding RNA molecules with a myriad of functions still being determined. Among the non-coding RNAs, long non-coding RNAs (lncRNA) and enhancer RNAs (eRNA) are found to be most copious. While their exact biological functions and mechanisms of action are currently unknown, technologies such as next-generation RNA sequencing (RNA-seq) and global nuclear run-on sequencing (GRO-seq) have begun deciphering their expression patterns and biological significance. In addition to their identification, it has been shown that the expression of long non-coding RNAs and enhancer RNAs can vary due to spatial, temporal, developmental, or hormonal variations. In this review, we explore newly reported information on estrogen-regulated eRNAs and lncRNAs and their associated biological functions to help outline their markedly prominent roles in estrogen-dependent signaling.

scholarly journals Comparative analysis of RNA enrichment methods for preparation of Cryptococcus neoformans RNA sequencing libraries

2021 ◽  
Author(s):  
Calla L. Telzrow ◽  
Paul J. Zwack ◽  
Shannon Esher Righi ◽  
Fred S. Dietrich ◽  
Cliburn Chan ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Rna Sequencing ◽  
Rnase H ◽  
Rrna Genes ◽  
Rna Seq ◽  
Protein Coding ◽  
Expression Levels ◽  
Non Coding Rna ◽  
Long Non Coding Rna ◽  
Enrichment Methods

ABSTRACTRibosomal RNA (rRNA) is the major RNA constituent of cells, therefore most RNA sequencing (RNA-Seq) experiments involve removal of rRNA. This process, called RNA enrichment, is done primarily to reduce cost: without rRNA removal, deeper sequencing would need to be performed to balance the sequencing reads wasted on rRNA. The ideal RNA enrichment method would remove all rRNA without affecting other RNA in the sample. We have tested the performance of three RNA enrichment methods on RNA isolated from Cryptococcus neoformans, a fungal pathogen of humans. We show that the RNase H depletion method unambiguously outperforms the commonly used Poly(A) isolation method: the RNase H method more efficiently depletes rRNA while more accurately recapitulating the expression levels of other RNA observed in an unenriched “gold standard”. The RNase H depletion method is also superior to the Ribo-Zero depletion method as measured by rRNA depletion efficiency and recapitulation of protein-coding gene expression levels, while the Ribo-Zero depletion method performs moderately better in preserving non-coding RNA (ncRNA). Finally, we have leveraged this dataset to identify novel long non-coding RNA (lncRNA) genes and to accurately map the C. neoformans mitochondrial rRNA genes.ARTICLE SUMMARYWe compare the efficacy of three different RNA enrichment methods for RNA-Seq in Cryptococcus neoformans: RNase H depletion, Ribo-Zero depletion, and Poly(A) isolation. We show that the RNase H depletion method, which is evaluated in C. neoformans samples for the first time here, is highly efficient and specific in removing rRNA. Additionally, using data generated through these analyses, we identify novel long non-coding RNA genes in C. neoformans. We conclude that RNase H depletion is an effective and reliable method for preparation of C. neoformans RNA-Seq libraries.

scholarly journals Differential Expression of Long Non-Coding RNA (lncRNA) in Mediterranean Mussel (Mytilus galloprovincialis) Hemocytes under Immune Stimuli

Genes ◽  
2021 ◽  
Vol 12 (9) ◽  
pp. 1393
Author(s):  
Patricia Pereiro ◽  
Rebeca Moreira ◽  
Beatriz Novoa ◽  
Antonio Figueras
Keyword(s):  
Regulation Of Gene Expression ◽  
Poly I:C ◽  
Rna Seq ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Discrete Response ◽  
Mediterranean Mussel ◽  
Long Non Coding Rna ◽  
Mollusk Species

The Mediterranean mussel is one of the most economically relevant bivalve mollusk species in Europe and China. The absence of massive mortalities and their resistance to pathogens affecting other cultured bivalves has been under study in recent years. The transcriptome response of this species to different immune stimuli has been extensively studied, and even the complexity of its genome, which has recently been sequenced, has been suggested as one of the factors contributing to this resistance. However, studies concerning the non-coding RNA profiles remain practically unexplored—especially those corresponding to the lncRNAs. To the best of our knowledge, this is the second characterization and study of lncRNAs in this bivalve species. In this work, we identified the potential repertoire of lncRNAs expressed in mussel hemocytes, and using RNA-Seq we analyzed the lncRNA profile of mussel hemocytes stimulated in vitro with three different immune stimuli: LPS, poly I:C, and β-glucans. Compared to unstimulated hemocytes, LPS induced the highest modulation of lncRNAs, whereas poly I:C and β-glucans induced a similar discrete response. Based on the potential cis-regulatory activity of the lncRNAs, we identified the neighboring protein-coding genes of the regulated lncRNAs to estimate—at least partially—the processes in which they are implicated. After applying correlation analyses, it seems that—especially for LPS—the lncRNAs could participate in the regulation of gene expression, and substantially contribute to the immune response.

Bone Marrow Microenvironment Regulates Alternative Splicing Events in Myeloma Cells through Downregulation of RNA Binding Protein Fox2

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4714-4714
Author(s):  
Weihua Song ◽  
Chaolin Zhang ◽  
Yiguo Hu ◽  
Maria Gkotzamanidou ◽  
Parantu Shah ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Alternative Splicing ◽  
Cell Lines ◽  
Actin Polymerization ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Splicing Factor ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Protein Coding

Abstract Alternative splicing is a crucial mechanism for gene regulation, which enhances the diversity of transcriptome and proteome. Misregulation of alternative splicing has been implicated in number of disease processes including cancer. Our data utilizing exon array profile from 170 uniformly treated newly diagnosed patients with MM confirms clinical relevance of splicing as demonstrated by impact of level and extent of alternate splicing on both progression free and overall survival. Fox2, a RNA splicing factor, is one of the most important genes predicting clinical outcome in these patients. We confirmed Fox2 expression in 10 MM cell lines at both RNA and protein levels. Immunohistochemistry staining showed a predominant nuclear localization of Fox2. Importantly, we also observed that MM cell - bone marrow stromal cells (BMSC) interaction led to significant inhibition of Fox2 expression in MM cells. Similar response was also observed using BMSC supernatants. While IL6 treatment significantly downregulated the expression of Fox2 in MM1S and RPMI8226 cells in a dose-dependent manner, IGF-1 treatment had no significant impact on Fox2 expression in MM cell lines. Since Fox2 has been described to plays a role in the maintenance of cell cytoskeleton, we therefore evaluated whether Fox2 might influence the migration and adhesion in MM cells. Transwell migration assay showed enhanced migration rate of Fox2-knocking down- MM1S and RPMI8226 cells versus controls. We also observed the increased cell adhesion to fibronetin in both cell lines upon Fox2 knockdown. Actin polymerization evaluated by Alexa488-conjugated phalloidin staining and confocal microscope analysis showed Fox2 knocking down cells with increased actin polymerization in both MM1S and RPMI8226 cell lines. Interstingly, we observed that Fox2 knockdown in MM cell lines did not affect the cell proliferation and survival. As Fox-2 is a splicing factor, we further evaluated the molecular impact of Fox2 expression in multiple myeloma by RNA-seq analysis. Our data revealed that Fox2 functions in regulating both protein-coding and non-coding RNA alternative splicing. Knockdown of Fox2 resulted in significant isoform up-regulation (60 in MM1S and 151 in RPMI8226) and down-regulation (70 in MM1S and 69 in RPMI8226). Gene enrichment analysis showed these genes are clustered in cell cytoskeleton regulation, microtubule-based movement, ATP binding, amongst others. Our study then focused on Fox2 knockdown-induced significant isoform switch in MM cell lines. We designed the primers testing the spliced exons and confirm the isoform switch in MM cells by PCR analysis (e.g. Pyk2 and PFDN6). Importantly, our RNA seq data showed that Fox2 regulates the expression of a series of microRNAs and long-noncoding RNAs (e.g. MALAT1 RPMI8226 CNT (58) vs Fox2 knockdown (108)), which provides us a new insight into impact of Fox2 on non-protein coding RNAs. We have also validated the RNA-seq data by Q-PCR analysis. In summary, our results identify Fox2 as a biologically important RNA binding protein that is regulated by bone marrow microenvironment interaction and with essential function and potential clinical implications in multiple myeloma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Insights into long non-coding RNA regulation of anthocyanin carrot root pigmentation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Constanza Chialva ◽  
Thomas Blein ◽  
Martin Crespi ◽  
Diego Lijavetzky
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Expression Profiles ◽  
Rna Seq ◽  
Rna Regulation ◽  
Protein Coding ◽  
New Genes ◽  
Non Coding Rna ◽  
Storage Organs ◽  
Anthocyanin Production ◽  
Long Non Coding Rna

AbstractCarrot (Daucus carota L.) is one of the most cultivated vegetable in the world and of great importance in the human diet. Its storage organs can accumulate large quantities of anthocyanins, metabolites that confer the purple pigmentation to carrot tissues and whose biosynthesis is well characterized. Long non-coding RNAs (lncRNAs) play critical roles in regulating gene expression of various biological processes in plants. In this study, we used a high throughput stranded RNA-seq to identify and analyze the expression profiles of lncRNAs in phloem and xylem root samples using two genotypes with a strong difference in anthocyanin production. We discovered and annotated 8484 new genes, including 2095 new protein-coding and 6373 non-coding transcripts. Moreover, we identified 639 differentially expressed lncRNAs between the phenotypically contrasted genotypes, including certain only detected in a particular tissue. We then established correlations between lncRNAs and anthocyanin biosynthesis genes in order to identify a molecular framework for the differential expression of the pathway between genotypes. A specific natural antisense transcript linked to the DcMYB7 key anthocyanin biosynthetic transcription factor suggested how the regulation of this pathway may have evolved between genotypes.

scholarly journals Non-Coding RNA Signatures of B-Cell Acute Lymphoblastic Leukemia

2021 ◽  
Author(s):  
Princess D. Rodriguez ◽  
Hana Paculova ◽  
Sophie Kogut ◽  
Jessica Heath ◽  
Hilde Schjerven ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Molecular Mechanisms ◽  
Lymphoblastic Leukemia ◽  
Transcriptome Profiling ◽  
Rna Seq ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Technological Developments ◽  
Cell Acute Lymphoblastic Leukemia

Non-coding RNAs (ncRNAs) comprise a diverse class of non-protein coding transcripts that regulate critical cellular processes associated with cancer. Advances in RNA-sequencing (RNA-Seq) have led to the characterization of non-coding RNA expression across different types of human cancers. Through comprehensive RNA-Seq profiling, a growing number of studies demonstrate that ncRNAs, including long non-coding RNA (lncRNAs) and microRNAs (miRNA), play central roles in progenitor B-cell Acute Lymphoblastic Leukemia (B-ALL) pathogenesis. Furthermore, due to their central roles in cellular homeostasis and their potential as biomarkers, the study of ncRNAs continues to provide new insight into the molecular mechanisms of B-ALL. This article reviews the ncRNA signatures reported for all B-ALL subtypes, focusing on technological developments in transcriptome profiling and recently discovered examples of ncRNAs with biologic and therapeutic relevance in B-ALL.

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