Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories

2013 ◽  
Vol 31 (11) ◽  
pp. 1015-1022 ◽  
Author(s):  
Peter A C 't Hoen ◽  
◽  
Marc R Friedländer ◽  
Jonas Almlöf ◽  
Michael Sammeth ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Throughput ◽  
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 Comprehensive assessment of multiple biases in small RNA sequencing reveals significant differences in the performance of widely used methods

2018 ◽  
Author(s):  
Carrie Wright ◽  
Anandita Rajpurohit ◽  
Emily E. Burke ◽  
Courtney Williams ◽  
Leonardo Collado-Torres ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
Reverse Transcription ◽  
High Throughput Sequencing ◽  
Small Rna Sequencing ◽  
Library Preparation ◽  
Adapter Ligation ◽  
Improve Accuracy ◽  
Reliable Quantification

ABSTRACTHigh-throughput sequencing offers advantages over other quantification methods for microRNA (miRNA), yet numerous biases make reliable quantification challenging. Previous evaluations of the biases associated with small RNA sequencing have focused on adapter ligation bias with limited evaluation of reverse transcription or amplification biases. Furthermore, evaluations of the accuracy of quantifications of isomiRs (miRNA isoforms) or the influence of starting amount on performance have been very limited and no study has yet evaluated differences in the quantification of isomiRs of altered length. In addition, no studies have yet compared the consistency of results derived from multiple moderate starting inputs. We therefore evaluated quantifications of miRNA and isomiRs using four library preparation kits, with various starting amounts, as well as quantifications following removal of duplicate reads using unique molecular identifiers (UMIs) to mitigate reverse transcription and amplification biases. All methods resulted in false isomiR detection; however, the adapter-free method tested was especially prone to false isomiR detection. We demonstrate that using UMIs improves accuracy and we provide a guide for input amounts to improve consistency. Our data show differences and limitations of current methods, thus raising concerns about the validity of quantification of miRNA and isomiRs across studies. We advocate for the use of UMIs to improve accuracy and reliability of miRNA quantifications.

scholarly journals High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq)

2017 ◽  
pp. 231-243 ◽  
Author(s):  
Eva Hrabeta-Robinson ◽  
Erin Marcus ◽  
Aaron E. Cozen ◽  
Eric M. Phizicky ◽  
Todd M. Lowe
Keyword(s):  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
Small Rna Sequencing

scholarly journals Erratum: Corrigendum: Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories

2014 ◽  
Vol 32 (3) ◽  
pp. 291-291
Author(s):  
Peter A C 't Hoen ◽  
◽  
Marc R Friedländer ◽  
Jonas Almlöf ◽  
Michael Sammeth ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
Small Rna Sequencing

High-throughput small RNA sequencing for evaluation of grapevine sanitation efficacy

2019 ◽  
Vol 267 ◽  
pp. 66-70 ◽  
Author(s):  
Ales Eichmeier ◽  
Marcela Kominkova ◽  
Jakub Pecenka ◽  
Petr Kominek
Keyword(s):  
Rna Sequencing ◽  
High Throughput ◽  
Small Rna ◽  
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.

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