ABSTRACT
In gene quantification and expression analysis, issues with sample selection and processing can be serious, as they can easily introduce irrelevant variables and lead to ambiguous results. This study aims to investigate the extent and mechanism of the impact of sample selection and processing on ribonucleic acid (RNA) sequencing. RNA from PBMCs and blood samples was investigated in this study. The integrity of this RNA was measured under different storage times. All the samples underwent high-throughput sequencing for comprehensive evaluation. The differentially expressed genes and their potential functions were analyzed after the samples were placed at room temperature for 0h, 4h and 8h, and different feature changes in these samples were also revealed. The sequencing results showed that the differences in gene expression were higher with an increased storage time, while the total number of genes detected did not change significantly. There were five genes showing gradient patterns over different storage times, all of which were protein-coding genes that had not been mentioned in previous studies. The effect of different storage times on seemingly the same samples was analyzed in this present study. This research, therefore, provides a theoretical basis for the long-term consideration of whether sample processing should be adequately addressed.
Subject(s)
High-Throughput Nucleotide Sequencing , RNA , Sequence Analysis, RNA , Humans , High-Throughput Nucleotide Sequencing/methods , Sequence Analysis, RNA/methods , RNA/genetics , RNA/blood , Leukocytes, Mononuclear/metabolism , Gene Expression Profiling/methods , Male , Specimen Handling/methods , Blood Specimen Collection/methods , FemaleABSTRACT
Parkinson's disease (PD) is the second most common neurodegenerative disease and although many studies have been done on this disease, the underlying mechanisms are still poorly understood and further studies are warranted. Therefore, this study identified circRNA expression profiles in the cerebral cortex (CC), hippocampus (HP), striatum (ST), and cerebellum (CB) regions of the 1-methyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model using RNA sequencing (RNA-seq), and differentially expressed circRNA were validated using reverse transcription quantitative real-time PCR (qRT-PCR). Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and competing endogenous RNA (ceRNA) network analyses were also performed to explore the potential function of circRNAs. The results show that, compared with the control group, 24, 66, 71, and 121 differentially expressed circRNAs (DE-circRNAs) were found in the CC, HP, ST, and CB, respectively. PDST vs. PDCB, PDST vs. PDHP, and PDCB vs. PDHP groups have 578, 110, and 749 DE-circRNAs, respectively. Then, seven DE-cirRNAs were selected for qRT-PCR verification, where the expressions were consistent with the sequencing analysis. The GO and KEGG pathway analyses revealed that these DE-circRNAs participate in several biological functions and signaling pathways, including glutamic synapse, neuron to neuron synapse, cell morphogenesis involved in neuron differentiation, Parkinson's disease, axon guidance, cGMP-PKG signaling pathway, and PI3K-Akt signaling pathway. Furthermore, the KEGG analysis of the target genes predicted by DE-circRNAs indicated that the target genes predicted by mmu_circRNA_0003292, mmu_circRNA_0001320, mmu_circRNA_0005976, and mmu_circRNA_0005388 were involved in the PD-related pathway. Overall, this is the first study on the expression profile of circRNAs in the different brain regions of PD mouse model. These results might facilitate our understanding of the potential roles of circRNAs in the pathogenesis of PD. Moreover, the results also indicate that the mmu_circRNA_0003292-miRNA-132-Nr4a2 pathway might be involved in the regulation of the molecular mechanism of Parkinson's disease.
Subject(s)
Brain/metabolism , Gene Expression Profiling , Parkinson Disease/genetics , RNA, Circular , Transcriptome , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/adverse effects , Animals , Computational Biology/methods , Disease Models, Animal , Gene Expression Profiling/methods , Gene Expression Regulation , Gene Ontology , Gene Regulatory Networks , Mice , Reproducibility of ResultsABSTRACT
DNA methylation is an important epigenetic marker that affects gene expression. Cell-free DNA methylation detection is a promising approach as abnormal distribution of DNA methylation is one of the hallmarks of many cancers and methylation changes occur early during carcinogenesis. This review summarizes the existing literature and reviews on the detection methods based on next generation sequencing for DNA methylation. The review also discusses the feasibility of detecting cfDNA methylation and the latest progress.
Subject(s)
Biomarkers, Tumor/analysis , Cell-Free Nucleic Acids/analysis , DNA Methylation , DNA, Neoplasm/analysis , Neoplasms/diagnosis , Biomarkers, Tumor/genetics , Cell-Free Nucleic Acids/genetics , DNA, Neoplasm/genetics , Early Detection of Cancer , High-Throughput Nucleotide Sequencing , Humans , Neoplasms/geneticsABSTRACT
MicroRNAs are a class of noncoding RNAs that can be involved in the regulation of gene expression in cancers, including lung cancer. Our previous research has shown that miR-486-5p is one of the most downregulated microRNAs in tissue and serum samples of lung cancer as a good diagnostic biomarker. The objective of this study is to investigate the roles of miR-486-5p in the progression of lung cancer. In this study, miR-486-5p was further validated to be significantly downregulated in additional nonsmall cell lung cancer (NSCLC) tissue, serum, and cell samples by quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the expression level of miR-486-5p was significantly associated with clinical phenotype of NSCLC. The PIK3R1 gene was confirmed to be a direct target of miR-486-5p by dual-luciferase reporter assay, and the expression level of miR-486-5p was inversely correlated with that of PIK3R1 in tumor tissues (r = -0.774, p < 0.01). Overexpressed miR-486-5p effectively inhibited cell proliferation and invasion and successfully induced apoptosis in vitro. PIK3R1 was involved in the suppression of miR-486-5p on cell growth. It can be concluded that miR-486-5p may act as a tumor suppressor contributing to the progression of NSCLC, and miR-486-5p would be a diagnostic and prognostic biomarker and a potential therapeutic target for lung cancer.
ABSTRACT
MicroRNAs (miRNAs) are a series of promising molecules that could regulate gene expression, while its expression level and type are strictly related to the early diagnosis, targeted therapy, and prognosis of diseases. Improved detection accuracy of miRNAs would lead to unprecedented progress in early treatment. Numerous methods to improve sensitivity have been proposed and confirmed valuable in miRNAs detection recently. However, the research which especially targets at improving detection specificity remains rare. This Feature gives a retrospective summary of the most common detection methods including Northern blot, reverse transcription quantitative polymerase chain reaction, next-generation sequencing, and microarray. Common methods play an important role in many aspects because of maturity and stability. However, the problem of specificity still exists. Then, this Feature summarizes breakthroughs in traditional techniques which are closely related to progressive methods to improve specificity. In addition, considerable studies focusing on improving accuracy are described in detail. Most of them involve the reasonable combination of common methods, and the strategies that contribute to improving accuracy are classified. This Feature aims to illustrate the importance of detection accuracy and explore how to improve specificity on detecting miRNAs, especially homologous families.
