A New Method for Quantitative Real-Time Polymerase Chain Reaction Data Analysis

Rao, Xiayu; Lai, Dejian; Huang, Xuelin

doi:10.1089/cmb.2012.0279

Cited by 961 publications

(1,013 citation statements)

References 45 publications

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“…The internal reference gene in this experiment was glyceraldehyde-phosphate dehydrogenase and the primer sequences of ZEB1-AS1 were 5′-CCGTGGGCACTGCTGAAT-3′ (forward) and 5′-CTGCTGGCAAGCGGAACT-3′ (reverse). The expression was calculated by using the 2 -ΔΔCt method [21]. The experiment was repeated 3 times to validate the result.…”

Section: Quantitative Real-time Polymerase Chain Reactionmentioning

confidence: 99%

Long Non-Coding RNA ZEB1-Antisense 1 Affects Cell Migration and Invasion of Cervical Cancer by Regulating Epithelial-Mesenchymal Transition via the p38MAPK Signaling Pathway

Gan¹,

Chen²,

Liu³

et al. 2018

Gynecol Obstet Invest

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Aim: To investigate whether long non-coding RNA (lncRNA) ZEB1 antisense 1 (ZEB1-AS1) affects cell migration and invasion of cervical cancer by regulating epithelial-mesenchymal transition (EMT) via the p38MAPK pathway. Methods: Human cervical cancer cell line Hela was classified into Control, NC siRNA, ZEB1-AS1 siRNA, SB203580 (p38MAPK pathway inhibitor) and ZEB1-AS1 siRNA + Anisomycin (p38MAPK pathway activator) groups. Quantitative real-time polymerase chain reaction was performed for ZEB1-AS1 expression, Western blotting to measure p38MAPK signaling pathway-/EMT-related proteins, and Wound-healing and Transwell assays to evaluate cell migration and invasion respectively. Results: ZEB1-AS1 was upregulated in cancer tissues and related to major clinicopathological features of cervical cancer. Besides, patients with lower-ZEB1-AS1-expression had a higher 5-year survival rate than those patients with higher-ZEB1-AS1-expression. High ZEB1-AS1 expression and advanced Federation of Gynecology and Obstetrics stage were independent risk factors for patients’ prognosis. Both ZEB1-AS1 siRNA and SB203580 effectively reduced p-p38 expression and the migration and invasion of Hela cells, with elevation of E-cadherin and reduction of Vimentin and N-cadherin. However, inhibitory effects of ZEB1-AS1 siRNA on EMT as well as cell migration and invasion of the Hela cell were reversed by Anisomycin. Conclusion: Inhibition of ZEB1-AS1 can block the p38MAPK signaling pathway, ultimately restricting the EMT and suppressing cell migration and invasion of cervical cancer cells.

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Section: Quantitative Real-time Polymerase Chain Reactionmentioning

confidence: 99%

Long Non-Coding RNA ZEB1-Antisense 1 Affects Cell Migration and Invasion of Cervical Cancer by Regulating Epithelial-Mesenchymal Transition via the p38MAPK Signaling Pathway

Gan¹,

Chen²,

Liu³

et al. 2018

Gynecol Obstet Invest

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“…This method relies on comparing the CT values of the target and reference samples, using a reference (endogenous housekeeping) gene as the normal izer. Finally, the method results in the fold change of target gene expression relative to a reference sample, normalized to a housekeeping gene (39).…”

Section: Transcriptomicsmentioning

confidence: 99%

The most common technologies and tools for functional genome analysis

Gasperskaja¹,

Kučinskas²

2017

AML

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Since the sequence of the human genome is complete, the main issue is how to understand the information written in the DNA se quence. Despite numerous genomewide studies that have already been performed, the challenge to determine the function of genes, gene products, and also their interaction is still open. As changes in the human genome are highly likely to cause pathological con ditions, functional analysis is vitally important for human health.For many years there have been a variety of technologies and tools used in functional genome analysis. However, only in the past decade there has been rapid revolutionizing progress and improvement in highthroughput methods, which are rang ing from traditional realtime polymerase chain reaction to more complex systems, such as nextgeneration sequencing or mass spectrometry. Furthermore, not only laboratory investigation, but also accurate bioinformatic analysis is required for reliable scientific results. These methods give an opportunity for accu rate and comprehensive functional analysis that involves various fields of studies: genomics, epigenomics, proteomics, and inter actomics. This is essential for filling the gaps in the knowledge about dynamic biological processes at both cellular and organis mal level. However, each method has both advantages and limita tions that should be taken into account before choosing the right method for particular research in order to ensure successful study. For this reason, the present review paper aims to describe the most frequent and widelyused methods for the comprehen sive functional analysis.

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“…A LightCycler 480 system was used for a fluorescence quantitative PCR reaction with the following reaction conditions: pre-denaturation at 95°C for 10 min, denaturation at 94°C for 15 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min, with 40 cycles. With U6 as an internal reference, the relative mRNA expression was calculated by the 2 -△△CT method [35].…”

Section: Quantitative Real-time Polymerase Chain Reaction (Qrt-pcr)mentioning

confidence: 99%

Downregulation of MicroRNA-330 Correlates with the Radiation Sensitivity and Prognosis of Patients with Brain Metastasis from Lung Cancer

Jiang

Zhu²,

Zhang

et al. 2017

Cell Physiol Biochem

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Background: The present study sought to explore the role of in predicting the radiation response and prognosis of patients with brain metastasis (BM) from lung cancer (LC). Methods: Patients with BM from LC were identified and classified into radiation-sensitive and radiation-resistant groups according to the overall survival rate, local and distant recurrence rate after conventional whole-brain radiation therapy. Quantitative realtime polymerase chain reaction (qRT-PCR) was used to detect miR-330 expression in serum. Receiver operating characteristic (ROC) curves were used to evaluate the prognostic value of miR-330 for the radiation sensitivity of brain metastasis from LC. Related clinical factors for radiation sensitivity were assessed by logistic regression analysis, and a survival analysis was conducted using COX regression and the Kaplan-Meier method. Results: MiR-330 exhibited lower expression in the radiation-sensitive group than in the radiation-resistant group. The area under the ROC curve of miR-330 for predicting radiation sensitivity was 0.898 (optimal cut-off value, 0.815), with a sensitivity of 71.7% and a specificity of 90.1%. After radiation therapy, patients with low miR-330 expression, compared to patients with high miR-330 expression, displayed a lower survival rate and a median survival time. MiR-330 expression was correlated with extracranial metastasis, maximum BM diameter, tumor-node-metastasis (TNM) stage and node (N) stage. Logistic regression and COX regression analyses revealed that extracranial metastasis, TNM stage, N stage and miR-330 expression were factors that influenced both radiation sensitivity and individual prognostic factors in patients with BM from LC. Conclusions: These findings indicate that the downregulation of miR-330 correlates with radiation sensitivity and poor prognosis in patients with BM from LC.

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A New Method for Quantitative Real-Time Polymerase Chain Reaction Data Analysis

Cited by 961 publications

References 45 publications

Long Non-Coding RNA ZEB1-Antisense 1 Affects Cell Migration and Invasion of Cervical Cancer by Regulating Epithelial-Mesenchymal Transition via the p38MAPK Signaling Pathway

Long Non-Coding RNA ZEB1-Antisense 1 Affects Cell Migration and Invasion of Cervical Cancer by Regulating Epithelial-Mesenchymal Transition via the p38MAPK Signaling Pathway

The most common technologies and tools for functional genome analysis

Downregulation of MicroRNA-330 Correlates with the Radiation Sensitivity and Prognosis of Patients with Brain Metastasis from Lung Cancer

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