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 miRA: adaptable novel miRNA identification in plants using small RNA sequencing data

2015 ◽  
Vol 16 (1) ◽  
Author(s):  
Maurits Evers ◽  
Michael Huttner ◽  
Anne Dueck ◽  
Gunter Meister ◽  
Julia C. Engelmann
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rna Sequencing ◽  
Sequencing Data ◽  
Novel Mirna ◽  
Mirna Identification

scholarly journals Comprehensive MicroRNA Expression Profile of the Mammary Gland in Lactating Dairy Cows With Extremely Different Milk Protein and Fat Percentages

2020 ◽  
Vol 11 ◽  
Author(s):  
Xiaogang Cui ◽  
Shengli Zhang ◽  
Qin Zhang ◽  
Xiangyu Guo ◽  
Changxin Wu ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Rna Sequencing ◽  
Differentially Expressed Genes ◽  
Small Rna ◽  
Milk Protein ◽  
Milk Composition ◽  
Differentially Expressed ◽  
Small Rna Sequencing ◽  
Rna Seq

A total of 31 differentially expressed genes in the mammary glands were identified in our previous study using RNA sequencing (RNA-Seq), for lactating cows with extremely high and low milk protein and fat percentages. To determine the regulation of milk composition traits, we herein investigated the expression profiles of microRNA (miRNA) using small RNA sequencing based on the same samples as in the previous RNA-Seq experiment. A total of 497 known miRNAs (miRBase, release 22.1) and 49 novel miRNAs among the reads were identified. Among these miRNAs, 71 were found differentially expressed between the high and low groups (p < 0.05, q < 0.05). Furthermore, 21 of the differentially expressed genes reported in our previous RNA-Seq study were predicted as target genes for some of the 71 miRNAs. Gene ontology and KEGG pathway analyses showed that these targets were enriched for functions such as metabolism of protein and fat, and development of mammary gland, which indicating the critical role of these miRNAs in regulating the formation of milk protein and fat. With dual luciferase report assay, we further validated the regulatory role of 7 differentially expressed miRNAs through interaction with the specific sequences in 3′UTR of the targets. In conclusion, the current study investigated the complexity of the mammary gland transcriptome in dairy cattle using small RNA-seq. Comprehensive analysis of differential miRNAs expression and the data from previous study RNA-seq provided the opportunity to identify the key candidate genes for milk composition traits.

scholarly journals Novel miRNA identification and comparative profiling of miRNA regulations revealed important pathways in Jinding duck ovaries by small RNA sequencing

3 Biotech ◽  
2020 ◽  
Vol 10 (2) ◽  
Author(s):  
Chaowu Yang ◽  
Xia Xiong ◽  
Xiaosong Jiang ◽  
Huarui Du ◽  
Qingyun Li ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rna Sequencing ◽  
Novel Mirna ◽  
Mirna Identification

scholarly journals Small RNA-sequencing for Analysis of Circulating miRNAs: Benchmark Study

2021 ◽  
Author(s):  
Peter Androvic ◽  
Sarka Benesova ◽  
Eva Rohlova ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Circulating Mirnas ◽  
Contributing Factors ◽  
Circulating Micrornas ◽  
Quantification Accuracy ◽  
Biomarker Research ◽  
Spurious Results

Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated development of several novel methods. Here, we present comparison of all small RNA-Seq library preparation approaches that are commercially available for quantification of miRNAs in biofluids. Using synthetic and human plasma samples, we compared performance of traditional two-adaptor ligation protocols (Lexogen, Norgen) as well as methods using randomized adaptors (NEXTflex), polyadenylation (SMARTer), circularization (RealSeq), capture probes (EdgeSeq) or unique molecular identifiers, UMIs (QIAseq). Globally, there was no single protocol outperforming others across all metrics. We documented limited overlap of measured miRNA profiles between methods largely owing to protocol-specific biases. We found that methods designed to minimize bias largely differ in their performance and we identified contributing factors. We found that usage of UMIs has rather negligible effect and if designed incorrectly can even introduce spurious results. Together, these results identify strengths and weaknesses of current methods and provide guidelines for applications of small RNA-Seq in biomarker research.

scholarly journals Characterization of Human Salivary Extracellular RNA by Next-generation Sequencing

2018 ◽  
Vol 64 (7) ◽  
pp. 1085-1095 ◽  
Author(s):  
Feng Li ◽  
Karolina Elżbieta Kaczor-Urbanowicz ◽  
Jie Sun ◽  
Blanca Majem ◽  
Hsien-Chun Lo ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Data Storage ◽  
Small Rna ◽  
Small Rnas ◽  
Rna Isolation ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Extracellular Rna ◽  
Library Construction ◽  
Rrna Depletion

Abstract BACKGROUND It was recently discovered that abundant and stable extracellular RNA (exRNA) species exist in bodily fluids. Saliva is an emerging biofluid for biomarker development for noninvasive detection and screening of local and systemic diseases. Use of RNA-Sequencing (RNA-Seq) to profile exRNA is rapidly growing; however, no single preparation and analysis protocol can be used for all biofluids. Specifically, RNA-Seq of saliva is particularly challenging owing to high abundance of bacterial contents and low abundance of salivary exRNA. Given the laborious procedures needed for RNA-Seq library construction, sequencing, data storage, and data analysis, saliva-specific and optimized protocols are essential. METHODS We compared different RNA isolation methods and library construction kits for long and small RNA sequencing. The role of ribosomal RNA (rRNA) depletion also was evaluated. RESULTS The miRNeasy Micro Kit (Qiagen) showed the highest total RNA yield (70.8 ng/mL cell-free saliva) and best small RNA recovery, and the NEBNext library preparation kits resulted in the highest number of detected human genes [5649–6813 at 1 reads per kilobase RNA per million mapped (RPKM)] and small RNAs [482–696 microRNAs (miRNAs) and 190–214 other small RNAs]. The proportion of human RNA-Seq reads was much higher in rRNA-depleted saliva samples (41%) than in samples without rRNA depletion (14%). In addition, the transfer RNA (tRNA)-derived RNA fragments (tRFs), a novel class of small RNAs, were highly abundant in human saliva, specifically tRF-4 (4%) and tRF-5 (15.25%). CONCLUSIONS Our results may help in selection of the best adapted methods of RNA isolation and small and long RNA library constructions for salivary exRNA studies.

scholarly journals Multiple Viral Infections Detected in Phytophthora condilina by Total and Small RNA Sequencing

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 620
Author(s):  
Leticia Botella ◽  
Thomas Jung
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Viral Infections ◽  
De Novo ◽  
Phytophthora Species ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Rt Pcr ◽  
Total Rna ◽  
Southern Portugal

Marine oomycetes have recently been shown to be concurrently infected by (−)ssRNA viruses of the order Bunyavirales. In this work, even higher virus variability was found in a single isolate of Phytophthora condilina, a recently described member of Phytophthora phylogenetic Clade 6a, which was isolated from brackish estuarine waters in southern Portugal. Using total and small RNA-seq the full RdRp of 13 different potential novel bunya-like viruses and two complete toti-like viruses were detected. All these viruses were successfully confirmed by reverse transcription polymerase chain reaction (RT-PCR) using total RNA as template, but complementarily one of the toti-like and five of the bunya-like viruses were confirmed when dsRNA was purified for RT-PCR. In our study, total RNA-seq was by far more efficient for de novo assembling of the virus sequencing but small RNA-seq showed higher read numbers for most viruses. Two main populations of small RNAs (21 nts and 25 nts-long) were identified, which were in accordance with other Phytophthora species. To the best of our knowledge, this is the first study using small RNA sequencing to identify viruses in Phytophthora spp.

scholarly journals Analysis of miRNAs Targeted Storage Regulatory Genes during Soybean Seed Development Based on Transcriptome Sequencing

Genes ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 408 ◽  
Author(s):  
Jing-Yao Yu ◽  
Zhan-Guo Zhang ◽  
Shi-Yu Huang ◽  
Xue Han ◽  
Xin-Yu Wang ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Seed Development ◽  
Small Rna ◽  
Target Genes ◽  
Soybean Seed ◽  
Regulatory Genes ◽  
Small Rna Sequencing ◽  
Grain Development ◽  
Regulatory Relationship ◽  
Novel Mirna

Soybeans are an important cash crop and are widely used as a source of vegetable protein and edible oil. MicroRNAs (miRNA) are endogenous small RNA that play an important regulatory role in the evolutionarily conserved system of gene expression. In this study, we selected four lines with extreme phenotypes, as well as high or low protein and oil content, from the chromosome segment substitution line (CSSL) constructed from suinong (SN14) and ZYD00006, and planted and sampled at three stages of grain development for small RNA sequencing and expression analysis. The sequencing results revealed the expression pattern of miRNA in the materials, and predicted miRNA-targeted regulatory genes, including 1967 pairs of corresponding relationships between known-miRNA and their target genes, as well as 597 pairs of corresponding relationships between novel-miRNA and their target genes. After screening and annotating genes that were targeted for regulation, five specific genes were identified to be differentially expressed during seed development and subsequently analyzed for their regulatory relationship with miRNAs. The expression pattern of the targeted gene was verified by Real-time Quantitative PCR (RT-qPCR). Our research provides more information about the miRNA regulatory network in soybeans and further identifies useful genes that regulate storage during soy grain development, providing a theoretical basis for the regulation of soybean quality traits.

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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