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Real-time quantitative PCR analysis of human plasma miRNA expresson

ABSTRACT: Plasma samples from 10 colonrectal cancer patients (CRCs) (including 5 stage II and 5 stage III patients) and 10 normal controls.An amount of 5 to 10 milliliters of whole blood were obtained from each participant.The plasma was obtained by centrifugation at 1200g for 10min at 4°C.To complete the removal of residual cellular components, plasma samples were recentrifuged at 12,000g for a further 10min at 4°C.A volume of 600μL of each plasma samples from CRC group or normal control group was picked out and uniformly mixed.

ORGANISM(S): Homo sapiens

PROVIDER: GSE38716 | GEO | 2012/12/29

SECONDARY ACCESSION(S): PRJNA168494

REPOSITORIES: GEO

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Project description:Purpose：Early diagnosis of nasopharyngeal carcinoma (NPC) is vital to improve the prognosis of these patients. However, early diagnosis of NPC is typically challenging.Therefore, we explored the roles and mechanisms of exosomes in plasma of patients with early-stage NPC. Methods: Exosomes in plasma were extracted by ultra-high-speed centrifugation.Western blot and transmission electron microscopy (TEM) were used to verify the purity of exosomes. The sequencing data (6 plasma samples from healthy volunteers vs. 6 NPC plasma samples) were analyzed by principal component analysis (PCA), DESeq2, gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG), and TargetScan. The differentially expressed miRNAs (DEmiRNAs) were obtained from the dataset (GSE118720) downloaded from the Gene Expression Omnibus (GEO) repository. Additionally, the datasets downloaded from the GEO database (GSE12452, GSE13597, GSE53819, GSE64634) were used to predict the target genes and functions of hsa-miR-1301-3p. qPCR was applied to verify the differences in the expressions of hsa-miR-1301-3p between 10 normal plasma and 10 NPC plasma samples. Results:Western blot, TEM, and Nanoparticle Tracking Analysis showed adequate purity of the extracted exosomes. RNA-seq analysis revealed 21 upregulated miRNAs, and 10 downregulated miRNAs in plasma exosomes of early-stage NPC patients. GO analysis showed that the target genes of DEmiRNAs were mainly enriched in DNA synthesis and transcription regulation. KEGG analysis revealed that DEmiRNAs were mainly enriched in PI3K-Akt and MAPK signaling pathways. Moreover, the expression of hsa-mir-1301-3p was verified to be significantly upregulated in enlarged samples of plasma exosomes. Conclusions:We identified several DEmiRNAs extracted from tumor-derived exosomes between normal plasma and early-stage NPC plasma. Bioinformatics analyses indicated that these DEmiRNAs may be related to NPC development. Our study may provide novel insights into underlying biomarkers and mechanisms of plasma exosomes in early-stage NPC.

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Real-time quantitative PCR analysis of human plasma miRNA expresson

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