Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)]

Reddy, Palakolanu Sudhakar; Dhaware, Mahamaya G.; Reddy, D. Srinivas; Reddy, Bommineni Pradeep; Divya, Kummari; Sharma, Kiran K.; Bhatnagar‐Mathur, Pooja

doi:10.1371/journal.pone.0205668

Cited by 14 publications

(5 citation statements)

References 60 publications

(69 reference statements)

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“…The LightCycle 480 system (Roche, Basel, Switzerland) was used to detect the expression level of exosomal miRNAs, the reaction system includes 2μl of cDNA template, 0.8μl of upstream and 0.8μl of downstream primers, 6.4μl of RNase-free water, and 10μl of SYBR-Green (Accurate Biotechnology, Hunan, China). U6 was used as an internal control ( 22 ). The relative gene expression was calculated using ΔCT (CT miRNAs -CT U6) as previously described ( 21 ).…”

Section: Methodsmentioning

confidence: 99%

Plasma exosomal miR-320d, miR-4479, and miR-6763-5p as diagnostic biomarkers in epithelial ovarian cancer

et al. 2022

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BackgroundExosomal miRNA had been proved as the promising biomarkers for multiple cancers including epithelial ovarian cancer (EOC). This study aimed to validate the diagnostic accuracy of exosomal miR-320d, miR-4479, and miR-6763-5p for EOC.Materials and methodsExosomes isolated from the plasma by ultracentrifugation were verified using TEM, qNano and western blot. MiRNAs sequencing was used to screen out the differential exosomal miRNAs and miR-320d, miR-4479, and miR-6763-5p were selected as candidates, which were further verified by RT-qPCR in 168 healthy donors and 161 primary EOC patients. Besides, the diagnostic accuracy of these three exosomal miRNAs were evaluated using the receiver operating characteristic curve (ROC).ResultsMiRNAs sequencing revealed 95 differential exosomal miRNAs between EOC patients and healthy donors. Subsequently, exosomal miR-320d, miR-4479, and miR-6763-5p were significantly down regulated in EOC patients compared with healthy controls and benign patients. More importantly, these three miRNAs could serve as circulating diagnostics biomarkers for EOC, possessing areas under the curve (AUC) of 0.6549, 0.7781, and 0.6834, respectively. Moreover, these three exosomal miRNAs levels were closely associated with lymph node metastasis, meanwhile exosomal miR-320d and miR-4479 expression was related to tumor stage.ConclusionExosomal miR-320d, miR-4479, and miR-6763-5p might serve as potential biomarkers for EOC.

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Section: Methodsmentioning

confidence: 99%

Plasma exosomal miR-320d, miR-4479, and miR-6763-5p as diagnostic biomarkers in epithelial ovarian cancer

et al. 2022

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“…qPCR was performed using TB-Green Premix Ex Taq II reagent (Takara Bio) on the LC480 (Roche Diagnostics, Germany). The relative gene expression levels were evaluated by the ΔCt method (Ct miRNA -Ct U6 ) as previously described ( 19 ). U6 was used as an internal control ( 20 ).…”

Section: Methodsmentioning

confidence: 99%

Tumor-Derived Exosomal miRNAs as Diagnostic Biomarkers in Non-Small Cell Lung Cancer

et al. 2020

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Background: Delayed diagnosis is the main obstacle to improve prognosis of non-small cell lung cancer (NSCLC). Novel biomarkers for the diagnosis of NSCLC are urgently needed. This study aimed to identify the specific exosomal miRNAs with diagnostic and prognostic potential in NSCLC patients. Materials and Methods: Transmission electron microscopy (TEM), qNano and western blots were used to characterize the exosomes isolated from the serum of NSCLC patients (n=330) and healthy donors (n=312) by ultracentrifugation. Exosomal miRNAs were profiled by miRNA microarrays and verified by quantitative PCR (qPCR). The diagnostic accuracy was determined by receiver operating characteristic (ROC) analysis. Results: A total of differential 22 miRNAs were screened out based on P < 0.05 and fold difference>2.0 by miRNA microarrays, among which, exosomal miR-5684 and miR-125b-5p were significantly down-regulated in NSCLC patients compared to healthy donors, processing favorable diagnostic efficiency for (early) NSCLC. Importantly, the exosomal miR-125b-5p were associated with metastasis (P < 0.0001), chemotherapeutic effect (P=0.007) and survival (P=0.008). Conclusion: Exosomal miR-5684 and miR-125b-5p levels are significantly downregulated in NSCLC patients, and serve as the promising diagnostic and prognostic biomarkers for NSCLC.

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“…The PCR reaction was evaluated by melting curve analysis. The relative quantification of miRNA expression was evaluated using the ΔCT method (Ct miRNA -Ct U6 ) as described previously (27).…”

Section: Methodsmentioning

confidence: 99%

Circulating Exosomal miR-150-5p and miR-99b-5p as Diagnostic Biomarkers for Colorectal Cancer

et al. 2019

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Background: Circulating exosomal miRNAs are potential non-invasive biomarkers for colorectal cancer. The present study aimed to validate the novel sensitive and specific exosomal miRNA biomarkers for diagnosing colorectal cancer (CRC).Patients and Methods: Exosomes isolated from the serum of CRC patients and healthy donors by ultracentrifugation were characterized using TEM, qNano, and immunoblotting. The exosomes from 2 healthy donors and 4 CRC patients were subjected to RNA isolation and miRNA sequencing. The differently expressed miRNAs from 165 primary CRC patients and 153 healthy donors were substantiated by RT-qPCR.Results: The RNA-sequence data analysis revealed that 29 exosomal miRNAs (20 downregulated and 9 upregulated) with >1.5-fold difference between CRC patients and healthy donors were selected. The serum exosomal miR-99b-5p and miR-150-5p levels were significantly downregulated in CRC patients as compared to healthy donors (p < 0.0001 and p < 0.0001, respectively) and benign disease (p = 0.009 and p < 0.0001, respectively). The expression levels of exosomal miR-99b-5p and miR-150-5p were significantly decreased in early CRC patients as compared to healthy donors (p < 0.0001 and p < 0.0001, respectively). The expression levels of exosomal miR-99b-5p and miR-150-5p were significantly increased postoperatively (p = 0.0058 and p < 0.0001, respectively).Conclusions: The present study demonstrated that serum exosomal miRNAs are promising, sensitive, specific, and non-invasive diagnostic biomarkers for CRC.Impact: This is the first study to specifically identify exosomal miR-99b-5p and miR-150-5p associated with CRC. This study, therefore, might deepen the understanding of tumor-derived exosomes for CRC diagnosis.

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Comprehensive evaluation of candidate reference genes for real-time quantitative PCR (RT-qPCR) data normalization in nutri-cereal finger millet [Eleusine Coracana (L.)]

Cited by 14 publications

References 60 publications

Plasma exosomal miR-320d, miR-4479, and miR-6763-5p as diagnostic biomarkers in epithelial ovarian cancer

Plasma exosomal miR-320d, miR-4479, and miR-6763-5p as diagnostic biomarkers in epithelial ovarian cancer

Tumor-Derived Exosomal miRNAs as Diagnostic Biomarkers in Non-Small Cell Lung Cancer

Circulating Exosomal miR-150-5p and miR-99b-5p as Diagnostic Biomarkers for Colorectal Cancer

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