NEXTflex™ Small RNA Sequencing for Small RNA Starting Material v1

protocols.io ◽  
2015 ◽  
Author(s):  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Starting Material ◽  
Small Rna Sequencing

Small RNA Sequencing to Discover Circulating MicroRNA Biomarkers of Testicular Toxicity in Dogs

2020 ◽  
pp. 109158182096151
Author(s):  
Jennifer C. Shing ◽  
Kai Schaefer ◽  
Shaun E. Grosskurth ◽  
Andy H. Vo ◽  
Tatiana Sharapova ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Exploratory Study ◽  
Quantitative Polymerase Chain Reaction ◽  
Small Rna Sequencing ◽  
Circulating Microrna ◽  
Testicular Toxicity ◽  
Potential Candidate ◽  
Drug Induced ◽  
Organ Systems

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.

scholarly journals Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 964
Author(s):  
Sarka Benesova ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
High Sensitivity ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Liquid Biopsies ◽  
Comprehensive Overview ◽  
Rna Molecules ◽  
Novel Mirna ◽  
The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

scholarly journals Identification of Zinc Deficiency-Responsive MicroRNAs in Brassica juncea Roots by Small RNA Sequencing

2013 ◽  
Vol 12 (11) ◽  
pp. 2036-2044 ◽  
Author(s):  
Dong-qing SHI ◽  
Yuan ZHANG ◽  
Jin-hu MA ◽  
Yu-long LI ◽  
Jin XU
Keyword(s):  
Rna Sequencing ◽  
Brassica Juncea ◽  
Zinc Deficiency ◽  
Small Rna ◽  
Small Rna Sequencing

Abstract 46: High-Density Lipoproteins Control Monocyte Differentiation Through microRNA Intercellular Communication

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Kasey C Vickers ◽  
Michael G Levin ◽  
Michael P Anderson ◽  
Qing Xu ◽  
Joshua Anzinger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
Cellular Response ◽  
Culture Media ◽  
Recipient Cell ◽  
Inflammatory Cells ◽  
High Density Lipoproteins ◽  
Small Rna Sequencing

Many HDL-microRNAs (miRNA) are well-characterized post-transcriptional regulators of inflammation, and are significantly increased on HDL with hypercholesterolemia and atherosclerosis in humans and mice. Therefore, we hypothesize that inflammatory cells uniquely control their own gene expression through cellular miRNA export to HDL and then regulate recipient cell gene expression through HDL-mediated miRNA delivery. To test this hypothesis, we used high-throughput proteomics, Open Arrays, small RNA sequencing, and gene expression microarrays. Human monocytes (plasma elutriation) were differentiated into dendritic cells and multiple macrophage phenotypes. Each cell-type was incubated with pure reconstituted HDL (rHDL), which was then purified from culture media by apolipoprotein A-I immunoprecipitation after 24 h, and both cellular and HDL-miRNAs were profiled using TaqMan Open Arrays. Macrophages were found to export high levels of miRNAs to HDL that inhibit monocyte/macrophage differentiation (miR-146a, miR-223); however, monocytes were also found to export many miRNAs associated with differentiation, including miR-92a, miR-222, miR-17, miR-20a, miR106a, and miR-21. Furthermore, many miRNAs were found to be transcribed in inflammatory cells, but completely exported to HDL and not retained in the cell. Most interestingly, HDL treatment was found to induce miR-223 transcription in monocytes, as determined by primary miR-223 transcript levels; however, intracellular levels of the mature form (miR-223) did not change. These results suggest that HDL induces the export of miRNAs it transports. PAR-CLIP with high-throughput small RNA sequencing was used to demonstrate that miRNAs are transferred from macrophages to endothelial cells and loaded onto cellular Argonaute 2-continaining RNA-induced silencing complexes. To demonstrate this in mice, human HDL, containing endogenous levels of miR-223, were injected into miR-223-null mice and inflammation-associated miRNA delivery was mapped in vivo. In summary, we found profound differences in the cellular response to HDL treatment and HDL-miRNA communication amongst inflammatory cell phenotypes that are physiologically relevant to cardiovascular disease.

scholarly journals Small RNA sequencing of sessile serrated polyps identifies microRNA profile associated with colon cancer

2018 ◽  
Vol 58 (1) ◽  
pp. 23-33 ◽  
Author(s):  
Priyanka Kanth ◽  
Mark W. Hazel ◽  
Kenneth M. Boucher ◽  
Zhihong Yang ◽  
Li Wang ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Rna Sequencing ◽  
Small Rna ◽  
Small Rna Sequencing ◽  
Serrated Polyps ◽  
Microrna Profile

scholarly journals Optimization of Extraction of Circulating RNAs from Plasma – Enabling Small RNA Sequencing

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e107259 ◽  
Author(s):  
Melanie Spornraft ◽  
Benedikt Kirchner ◽  
Bettina Haase ◽  
Vladimir Benes ◽  
Michael W. Pfaffl ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rna Sequencing

scholarly journals Analysis of conserved miRNAs in cynomolgus macaque genome using small RNA sequencing and homology searching

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9347
Author(s):  
Xia Huang ◽  
Shijia Li ◽  
Xiaoming Liu ◽  
Shuting Huang ◽  
Shuang Li ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Mirna Gene ◽  
Cynomolgus Macaque ◽  
Future Research ◽  
Small Rna Sequencing ◽  
Homology Searching ◽  
Macaque Genome ◽  
Cynomolgus Macaques ◽  
Mirna Clusters

MicroRNAs (miRNAs) are important regulators that fine-tune diverse cellular activities. Cynomolgus macaques (Macaca fascicularis) are used extensively in biomedical and pharmaceutical research; however, substantially fewer miRNAs have been identified in this species than in humans. Consequently, we investigated conserved miRNA profiles in cynomolgus macaques by homology searching and small RNA sequencing. In total, 1,455 high-confidence miRNA gene loci were identified, 408 of which were also confirmed by RNA sequencing, including 73 new miRNA loci reported in cynomolgus macaques for the first time. Comparing miRNA expression with age, we found a positive correlation between sequence conservation and expression levels during miRNA evolution. Additionally, we found that the miRNA gene locations in cynomolgus macaque genome were very flexible. Most were embedded in intergenic spaces or introns and clustered together. Several miRNAs were found in certain gene locations, including 64 exon-resident miRNAs, six splice-site-overlapping miRNAs (SO-miRNAs), and two pairs of distinct mirror miRNAs. We also identified 78 miRNA clusters, 68 of which were conserved in the human genome, including 10 large miRNA clusters predicted to regulate diverse developmental and cellular processes in cynomolgus macaque. Thus, this study not only expands the number of identified miRNAs in cynomolgus macaques but also provides clues for future research on the differences in miRNA repertoire between macaques and humans.

Sign in / Sign up

Export Citation Format

Share Document