scholarly journals Systematic assessment of commercially available low-input miRNA library preparation kits

2019 ◽  
Author(s):  
Fatima Heinicke ◽  
Xiangfu Zhong ◽  
Manuela Zucknick ◽  
Johannes Breidenbach ◽  
Arvind Sundaram ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Differentially Expressed ◽  
Detection Sensitivity ◽  
Library Preparation ◽  
Experimental Conditions ◽  
Total Rna ◽  
Systematic Assessment ◽  
Micro Rnas ◽  
Differentially Expressed Mirnas ◽  
Commercial Kits

AbstractHigh-throughput sequencing is increasingly favoured to assay the presence and abundance of micro RNAs (miRNAs) in biological samples, even from low RNA amounts, and a number of commercial vendors now offer kits that allow miRNA sequencing from sub-nanogram (ng) inputs. However, although biases introduced during library preparation have been documented, the relative performance of current reagent kits has not been investigated in detail. Here, six commercial kits capable of handling <100ng total RNA input were used for library preparation, performed by kit manufactures, on synthetic miRNAs of known quantities and human biological total RNA samples. We compared the performance of miRNA detection sensitivity, reliability, titration response and the ability to detect differentially expressed miRNAs. In addition, we assessed the use of unique molecular identifiers sequence (UMI) tags in one kit. We observed differences in detection sensitivity and ability to identify differentially expressed miRNAs between the kits, but none were able to detect the full repertoire of expected miRNAs. The reliability within the replicates of all kits was good, while larger differences were observed between the kits, although none could accurately quantify the majority of miRNAs. UMI tags, at least within the input ranges tested, offered little advantage to improve data utility. In conclusion, biases in miRNA abundance are heavily influenced by the kit used for library preparation, suggesting that comparisons of datasets prepared by different procedures should be made with caution. This article is intended to assist researchers select the most appropriate kit for their experimental conditions.

scholarly journals Serum tRNA-Derived Fragments as Potential Biomarkers in Children with Acute Intussusception

2020 ◽  
Author(s):  
Wei Chen ◽  
Lian Zhao ◽  
Wan-liang Guo
Keyword(s):  
High Throughput Sequencing ◽  
Predictive Accuracy ◽  
Predictive Biomarker ◽  
Differentially Expressed ◽  
Roc Curve Analysis ◽  
Potential Biomarker ◽  
Micro Rnas ◽  
Differentially Expressed Mirnas ◽  
Upregulated Genes ◽  
Selection Of

Abstract Background: The aim of the present study was to investigate whether transfer ribonucleic acid (tRNA)–derived fragments (tRFs) can serve as candidate biomarkers for pediatric intussusception.Methods: Using high-throughput sequencing technology, we identified differentially expressed tRFs, and ultimately selected three tRFs to establish a signature as a predictive biomarker of pediatric intussusception. Selection of these three upregulated genes was verified using quantitative reverse-transcription polymerase chain reaction (qRT-PCR). We conducted receiver operator characteristic (ROC) curve analysis to evaluate the predictive accuracy of the selected genes for pediatric intussusception.Results: We detected 732 tRFs and tRNA-derived stress-induced RNA (tiRNAs), 1705 micro-RNAs (miRNAs), 52 differentially expressed miRNAs, and 34 differentially expressed tRFs and tiRNAs between patients and controls. Compared with controls, we found 33 upregulated miRNAs, 24 upregulated tRFs and tiRNAs, 19 downregulated miRNAs, and 10 downregulated tRFs and tiRNAs in children with intussusception. Using qPCR, the expression trends of tRF-Leu-TAA-006, tRF-Gln-TTG-033 and tRF-Lys-TTT-028 were consistent with the sequencing results. AUCs of tRF-Leu-TAA-006, tRF-Gln-TTG-033 and tRF-Lys-TTT-028 were 0.984, 0.970 and 0.837, respectively.Conclusion: Circulating tRF-Leu-TAA-006, tRF-Gln-TTG-033 and tRF-Lys-TTT-028 expression might be a novel potential biomarker for diagnosis of pediatric intussusception.

scholarly journals Identification of MicroRNAs That Respond to Soybean Cyst Nematode Infection in Early Stages in Resistant and Susceptible Soybean Cultivars

2019 ◽  
Vol 20 (22) ◽  
pp. 5634 ◽  
Author(s):  
Piao Lei ◽  
Bing Han ◽  
Yuanyuan Wang ◽  
Xiaofeng Zhu ◽  
Yuanhu Xuan ◽  
...  
Keyword(s):  
Small Rna ◽  
Soybean Cyst Nematode ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Cyst Nematode ◽  
Differentially Expressed ◽  
Early Stages ◽  
Differentially Expressed Mirnas ◽  
Plant Pathogen Interactions ◽  
Insight Into

Soybean cyst nematode (SCN) causes heavy losses to soybean yield. In order to investigate the roles of soybean miRNAs during the early stages of infection (1 and 5 dpi), 24 small RNA libraries were constructed from SCN resistant cultivar Huipizhi (HPZ) and the susceptible Williams 82 (W82) cultivar for high-throughput sequencing. By sequencing the small RNA libraries, a total of 634 known miRNAs were identified, and 252 novel miRNAs were predicted. Altogether, 14 known miRNAs belonging to 13 families, and 26 novel miRNAs were differentially expressed and may respond to SCN infection in HPZ and W82. Similar expression results were also confirmed by qRT-PCR. Further analysis of the biological processes that these potential target genes of differentially expressed miRNAs regulate found that they may be strongly related to plant–pathogen interactions. Overall, soybean miRNAs experience profound changes in early stages of SCN infection in both HPZ and W82. The findings of this study can provide insight into miRNAome changes in both HPZ and W82 at the early stages of infection, and may provide a stepping stone for future SCN management.

scholarly journals Identification and study of differentially expressed miRNAs in aged NAFLD rats based on high-throughput sequencing

2020 ◽  
Vol 19 (3) ◽  
pp. 302-312 ◽  
Author(s):  
Ying Zhang ◽  
Danni Xiang ◽  
Xiaona Hu ◽  
Qingwei Ruan ◽  
Lina Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Differentially Expressed ◽  
Differentially Expressed Mirnas

scholarly journals Construction of miRNA-target networks using microRNA profiles of CVB3-infected HeLa cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hai Lan Yao ◽  
Mi Liu ◽  
Wen Jun Wang ◽  
Xin Ling Wang ◽  
Juan Song ◽  
...  
Keyword(s):  
Hela Cells ◽  
Regulatory Networks ◽  
Cellular Differentiation ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Expression Profiles ◽  
Differentially Expressed ◽  
Mirna Regulation ◽  
Cvb3 Infection ◽  
Differentially Expressed Mirnas

AbstractMicroRNAs (miRNAs) play an important role in regulating gene expression in multiple biological processes and diseases. Thus, to understand changes in miRNA during CVB3 infection, specific miRNA expression profiles were investigated at 3 h, 6 h, and 9 h postinfection in HeLa cells by small-RNA high-throughput sequencing. Biological implications of 68 differentially expressed miRNAs were analyzed through GO and KEGG pathways. Interaction networks between 34 known highly differentially expressed miRNAs and targets were constructed by mirDIP and Navigator. The predicted targets showed that FAM135A, IKZF2, PLAG1, ZNF148, PHC3, LCOR and DYRK1A, which are associated with cellular differentiation and transcriptional regulation, were recognized by 8 miRNAs or 9 miRNAs through interactional regulatory networks. Seven target genes were confirmed by RT-qPCR. The results showed that the expression of DYRK1A, FAM135A, PLAG1, ZNF148, and PHC3 were obviously inhibited at 3 h, 6 h, and 9 h postinfection. The expression of LCOR did not show a significant change, and the expression of IKZF2 increased gradually with prolonged infection time. Our findings improve the understanding of the pathogenic mechanism of CVB3 infection on cellular differentiation and development through miRNA regulation, which has implications for interventional approaches to CVB3-infection therapy. Our results also provide a new method for screening target genes of microRNA regulation in virus-infected cells.

scholarly journals Integrated analysis of miRNA and mRNA expression profiles in testes of Duroc and Meishan boars

2019 ◽  
Author(s):  
Haisheng Ding ◽  
Min Liu ◽  
Changfan Zhou ◽  
Xiangbin You ◽  
Tao Su ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Target Genes ◽  
Expression Profiles ◽  
Male Gamete ◽  
Mirna Expression Profile ◽  
Mapk Signaling ◽  
Differentially Expressed ◽  
Integrated Analysis ◽  
Testis Development ◽  
Differentially Expressed Mirnas

Abstract Background: MicroRNAs (miRNAs) are small non-coding RNAs playing vital roles in regulating posttranscriptional gene expression. Elucidating the expression regulation of miRNAs underlying pig testis development will contribute to a better understanding of boar fertility and spermatogenesis. Results: In this study, miRNA expression profile was investigated in testes of Duroc and Meishan boars at 20, 75, and 270 days of age by high-throughput sequencing. Forty-five differentially expressed miRNAs were identified from testes of Duroc and Meishan boars before and after puberty. Integrated analysis of miRNA and mRNA profiles predicted many miRNA-mRNA pairs. Gene ontology and biological pathway analyses revealed that predicted target genes of ssc-mir-423-5p, ssc-mir-34c, ssc-mir-107, ssc-165 mir-196b-5p, ssc-mir-92a, ssc-mir-320, ssc-mir-10a-5p, and ssc-mir-181b were involved in sexual reproduction, male gamete generation, and spermatogenesis, and GnRH, Wnt, and MAPK signaling pathway. Four significantly differentially expressed miRNAs and their predicted target genes were validated by quantitative real-time polymerase chain reaction, and phospholipase C beta 1 ( PLCβ1) gene was verified to be a target of ssc-mir-423-5p . Conclusions: This study provides an insight into the functional roles of miRNAs in testis development and spermatogenesis and offers useful resources for understanding differences in sexual function development caused by the change in miRNAs expression between Duroc and Meishan boars.

scholarly journals High-Throughput Sequencing Identification of Differentially Expressed microRNAs in Metastatic Ovarian Cancer with experimental validations

2020 ◽  
Author(s):  
Yang Gu ◽  
Shulan Zhang
Keyword(s):  
Ovarian Cancer ◽  
High Throughput Sequencing ◽  
Human Cancer ◽  
Quantitative Polymerase Chain Reaction ◽  
Gynecological Cancer ◽  
Metastatic Potential ◽  
Differentially Expressed ◽  
Luciferase Reporter ◽  
Downstream Target ◽  
Differentially Expressed Mirnas

Abstract Background: Ovarian cancer (OC) is a common gynecological cancer and characterized by high metastatic potential. MicroRNAs (miRNAs, miRs) have the promise to be harnessed as prognostic and therapeutic biomarkers for OC. Herein, we sought to identify differentially expressed miRNAs and mRNAs in metastatic OC, and to validate them with functional experiments. Methods: Differentially expressed miRNAs and miRNAs were screened from six pairs of primary OC tissues and metastatic tissues using an miRStar™ Human Cancer Focus miRNA & Target mRNA PCR Array. Then, gene expression profiling results were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot assays. The binding affinity between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. Expression of miR-7-5p and TGFβ2 was manipulated to assess their roles in malignant phenotypes of highly metastatic HO-8910PM cells. Results: MiRNA profiling and sequencing identified 12 miRNAs and 10 mRNAs that were differentially expressed in metastatic tissues. Gene ontology and Pathway analyses determined that 3 differentially expressed mRNAs (ITGB3, TGFβ2 and TNC) were related to OC metastasis. The results of RT-qPCR confirmed that the decrease of miR-7-5p was most significant in OC metastasis, while TGFβ2 was up-regulated in OC metastasis. Moreover, miR-7-5p targeted and negatively regulated TGFβ2. MiR-7-5p overexpression accelerated HO-8910PM cell viability and invasion, and TGFβ2 overexpression reversed the results. Meanwhile, simultaneous miR-7-5p and TGFβ2 overexpression rescued the cell activities. Conclusions: This study characterizes differentially expressed miRNAs and mRNAs in metastatic OC, where miR-7-5p and its downstream target were most closely associated with metastatic OC. Overexpression of miR-7-5p targets and inhibits TGFβ2 expression, thereby inhibiting the growth and metastasis of OC.

scholarly journals Immune-related miRNA-mRNA regulation network in the livers of DHAV-3-infected ducklings

2019 ◽  
Author(s):  
Fengyao Wu ◽  
Fengying Lu ◽  
Xin Fan ◽  
Jin Chao ◽  
Chuanmin Liu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
High Throughput Sequencing ◽  
Expression Patterns ◽  
Differentially Expressed ◽  
Receptor Interaction ◽  
Integrated Analysis ◽  
Mrna Regulation ◽  
Duck Hepatitis ◽  
Differentially Expressed Mirnas ◽  
Regulation Network

Abstract Background: Duck hepatitis A virus type 3 (DHAV-3) is one of the most harmful pathogens in the duck industry. However, the molecular mechanism underlying DHAV-3 infection in ducklings is remain poorly understood. To elucidate the genetic regulatory network for miRNA-mRNA and the signaling pathways involved in DHAV-3 infection in ducklings, we conducted global miRNA and mRNA expression profiling of duckling liver tissues infected with lethal DHAV-3 using high-throughput sequencing. Results: We found 156 differentially expressed miRNAs (DEMs) and 7717 differentially expressed miRNAs (DEGs) between mock-infected and DHAV-3-infected duckling livers. A total of 19,606 miRNA-mRNA pairs with negatively correlated expression patterns were identified in miRNA-mRNA networks constructed on the basis of these DEMs and DEGs. Moreover, immune-related pathways including the cytokine-cytokine receptor interaction, apoptosis, Toll-like receptor, Jak-STAT, and RIG-I-like receptor signaling pathway were significantly enriched through analyzing functions of mRNAs in the network in response to DHAV-3 infection. Besides, apl-miR-32-5p, apl-miR-125-5p, apl-miR-128-3p, apl-miR-460-5p, and novel-m0012-3p were identified as potential regulators in the immune-related signaling pathways during the DHAV-3 infection. Conclusions: To our knowledge, this is the first report on integrated analysis of miRNA-seq and mRNA-seq in DHAV-3-infected ducklings. The results indicated the important roles of miRNAs in regulating immune response genes and revealed the immune related miRNA-mRNA regulation network in the DHAV-3-infected duckling liver. Our findings may provide valuable information to further investigate the roles of miRNAs and their target genes in DHAV-3 replication and pathogenesis; additionally, they may offer clues for further understanding host-virus interactions.

scholarly journals Identification and characterization of heat-responsive microRNAs at the booting stage in two rice varieties 9311 and Nagina 22

Genome ◽  
2021 ◽  
Author(s):  
Ying Luo ◽  
Tao Wang ◽  
Dan Yang ◽  
Biao Luo ◽  
Weiping Wang ◽  
...  
Keyword(s):  
Stress Responses ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Elevated Temperatures ◽  
Differentially Expressed ◽  
Control Group ◽  
Microrna Target ◽  
Rice Varieties ◽  
Booting Stage ◽  
Differentially Expressed Mirnas

Abstract: MicroRNAs (miRNAs) are small, non-coding, regulatory RNAs that play important roles in abiotic stress responses in plants. but their regulatory roles in the adaptive response to heat stress at the booting stage in two rice varieties 9311 and Nagina 22, remain largely unknown. In this study, 464 known miRNAs and 123 potential novel miRNAs were identified. Of these miRNAs, a total of 90 differential expressed miRNAs were obtained with 9311 libraries as control group, of which 54 upregulated and 36 downregulated miRNAs. To gain insight into functional significance, 2773 potential target genes of these 90 differentially expressed miRNAs were predicted. GO enrichment showed that the predicted target genes of differentially expressed miRNAs including NACs, LACs, CSD, and Hsp40. KEGG pathway analysis showed that target genes of these differentially expressed miRNAs were significantly enriched in plant hormone signal transduction pathway. The expression levels of ten differentially expressed miRNAs and their target genes obtained by qRT-PCR were largely consistent with the sequencing results. This study lays a foundation for the elucidation of the miRNA-mediated regulatory mechanism in rice at elevated temperatures. Key words: rice, heat-responsive, microRNA, target gene, booting stage, high-throughput sequencing

scholarly journals Integrated analysis of miRNA and mRNA expression profiles in testes of Duroc and Meishan boars

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Haisheng Ding ◽  
Min Liu ◽  
Changfan Zhou ◽  
Xiangbin You ◽  
Tao Su ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Target Genes ◽  
Expression Profiles ◽  
Male Gamete ◽  
Mirna Expression Profile ◽  
Mapk Signaling ◽  
Differentially Expressed ◽  
Integrated Analysis ◽  
Testis Development ◽  
Differentially Expressed Mirnas

Abstract Background MicroRNAs (miRNAs) are small non-coding RNAs playing vital roles in regulating posttranscriptional gene expression. Elucidating the expression regulation of miRNAs underlying pig testis development will contribute to a better understanding of boar fertility and spermatogenesis. Results In this study, miRNA expression profile was investigated in testes of Duroc and Meishan boars at 20, 75, and 270 days of age by high-throughput sequencing. Forty-five differentially expressed miRNAs were identified from testes of Duroc and Meishan boars before and after puberty. Integrated analysis of miRNA and mRNA profiles predicted many miRNA-mRNA pairs. Gene ontology and biological pathway analyses revealed that predicted target genes of ssc-mir-423-5p, ssc-mir-34c, ssc-mir-107, ssc-mir-196b-5p, ssc-mir-92a, ssc-mir-320, ssc-mir-10a-5p, and ssc-mir-181b were involved in sexual reproduction, male gamete generation, and spermatogenesis, and GnRH, Wnt, and MAPK signaling pathway. Four significantly differentially expressed miRNAs and their predicted target genes were validated by quantitative real-time polymerase chain reaction, and phospholipase C beta 1 (PLCβ1) gene was verified to be a target of ssc-mir-423-5p. Conclusions This study provides an insight into the functional roles of miRNAs in testis development and spermatogenesis and offers useful resources for understanding differences in sexual function development caused by the change in miRNAs expression between Duroc and Meishan boars.

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