Data Normalization Strategies for MicroRNA Quantification

Schwarzenbach, Heidi; Silva, Andreia; Calin, George A.; Pantel, Klaus

doi:10.1373/clinchem.2015.239459

Cited by 399 publications

(394 citation statements)

References 90 publications

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“…The choice of a reference gene remains problematic and can have a serious impact on the actual available transcript levels and, consequently, on the biological interpretation of data (44 ). Although several groups have been focused on studying the expression levels of circulating miRNAs in plasma, it is remarkable that limited overlap has been observed between the findings of even very similar studies of the same disease.…”

Section: Discussionmentioning

confidence: 99%

Direct Comparison of Metastasis-Related miRNAs Expression Levels in Circulating Tumor Cells, Corresponding Plasma, and Primary Tumors of Breast Cancer Patients

et al. 2016

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BACKGROUND Circulating tumor cells (CTCs) and microRNAs (miRNAs) are important in liquid biopsies in which peripheral blood is used to characterize the evolution of solid tumors. We evaluated the expression levels of miR-21, miR-146a, miR-200c, and miR-210 in CTCs of breast cancer patients with verified metastasis and compared their expression levels in corresponding plasma and primary tumors. METHODS Expression levels of the miRNAs were quantified by quantitative reverse transcription PCR (RT-qPCR) in (a) 89 primary breast tumors and 30 noncancerous breast tissues and (b) CTCs and corresponding plasma of 55 patients with metastatic breast cancer and 20 healthy donors. For 30 of these patients, CTCs, corresponding plasma, and primary tumor tissues were available. RESULTS In formalin-fixed, paraffin-embedded tissues, these miRNAs were differentially expressed between primary breast tumors and noncancerous breast tissues. miR-21 (P < 0.001) and miR-146a (P = 0.001) were overexpressed, whereas miR-200c (P = 0.004) and miR-210 (P = 0.002) were underexpressed. In multivariate analysis, miR-146a overexpression was significantly [hazard ratio 2.969 (1.231–7.157), P = 0.015] associated with progression-free survival. In peripheral blood, all miRNAs studied were overexpressed in both CTC and corresponding plasma. There was a significant association between miR-21 expression levels in CTCs and plasma for 36 of 55 samples (P = 0.008). In plasma, ROC curve analysis revealed that miR-21, miR-146a, and miR-210 could discriminate patients from healthy individuals. CONCLUSIONS Metastasis-related miRNAs are overexpressed in CTCs and corresponding plasma; miR-21 expression levels highly correlate in CTCs and plasma; and miR-21, miR-146a, and miR-210 are valuable plasma biomarkers for discriminating patients from healthy individuals.

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Section: Discussionmentioning

confidence: 99%

Direct Comparison of Metastasis-Related miRNAs Expression Levels in Circulating Tumor Cells, Corresponding Plasma, and Primary Tumors of Breast Cancer Patients

et al. 2016

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“…A reliable expression analysis of miRNAs, particularly extracellular miRNAs, in bodily fluids still remains an analytical challenge because of the unique characteristics of miRNA molecules (small size, high homology among miRNAs within the same family and low concentrations in body fluids), the lack of standardized methodologies, and the different detection methods and sensitivities of the various commercial reagent kits and systems [73][74][75]. Currently, the methods most commonly used for the analysis of miRNA expression include RT-qPCR, microarrays and next-generation sequencing (NGS).…”

Section: Methods Of Mirna Expression Analysismentioning

confidence: 99%

miRNAs as Biomarkers and Therapeutic Targets in Non-Small Cell Lung Cancer: Current Perspectives

Florczuk¹,

Szpechcinski²,

Chorostowska‐Wynimko³

2017

Targ Oncol

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Lung cancer is the most common cancer worldwide. Up to 85% of lung cancer cases are diagnosed as nonsmall cell lung cancer (NSCLC). The effectiveness of NSCLC treatment is expected to be improved through the implementation of robust and specific biomarkers. MicroRNAs (miRNAs) are small, non-coding molecules that play a key role in the regulation of basic cellular processes, including differentiation, proliferation and apoptosis, by controlling gene expression at the post-transcriptional level.

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“…To avoid a false interpretation of data, the choice of endogenous and exogenous reference controls is crucial. RNU6, miR-93 and miR-16 are the most frequently used endogenous controls, to quantify miRNAs [19]. Four lncRNAs (BC200, Zfhx2as, H19 upstream conserved 1 and 2, and HOXA6as) have been shown to be suitable as normalizers for lncRNA levels [20].…”

Section: Techniques Applied For the Quantification Of Ncrnasmentioning

confidence: 99%

“…Four lncRNAs (BC200, Zfhx2as, H19 upstream conserved 1 and 2, and HOXA6as) have been shown to be suitable as normalizers for lncRNA levels [20]. For the inter-individual variability of an efficient handling, exogenous cel-miR-39 is usually spiked in the plasma/serum samples, prior to the quantification of ncRNAs [19].…”

Section: Techniques Applied For the Quantification Of Ncrnasmentioning

confidence: 99%

“…These findings show that the expression of miR-16 may be cancer-and stage-specifically regulated. On the other hand, the use of miR-16 as a normalizer may be a standard approach for most analyses, because it has been described as being highly expressed in plasma or serum of diverse cancer types, and relatively invariant across diverse blood samples [19].…”

Section: Diagnostic Value Of Cell-free Mirnas In Plasma and Serummentioning

confidence: 99%

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Diagnostic relevance of circulating cell-free and exosomal microRNAs and long non-coding RNAs in blood of cancer patients

Schwarzenbach

2016

LaboratoriumsMedizin

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MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) belong to a large group of non-coding RNA (ncRNA) molecules that possess diverse functions in epigenetic and chromatin structure modifications. In particular, they post-transcriptionally inhibit the protein expression of their target mRNAs and therefore, are involved in different tumor-associated signaling pathways. As the expression of both ncRNAs is frequently dysregulated in cancer and shows unique signatures, they may play a crucial role in cancer pathogenesis. They are passively and actively released into the blood circulation, and accordingly, their plasma/serum and exosomal profiles reflect disease development, tumor load and malignant progression towards metastatic relapse. Considering the biology and nature of ncRNAs in the regulation of different cellular processes, they may have potential clinical utility as blood-based tumor markers and future therapeutic targets in the treatment of cancer patients. The present review focuses on the diagnostic value of cellfree and exosomal ncRNAs in plasma/serum of cancer patients and the challenges to establish them as future tumor markers.Keywords: cell-free miRNAs; cell-free lncRNAs; exosomes; liquid biopsy.Zusammenfassung: MikroRNAs (miRNAs) und lange nicht-kodierende RNAs (lncRNAs) gehören zu einer großen Gruppe von nicht-kodierenden RNA-(ncRNA) Molekülen, die Funktionen bei epigenetischen und Chromatinstruktur-Modifikationen besitzen. Insbesondere inhibiieren sie post-transkriptionell die Proteinexpression ihrer Ziel-RNAs und sind deshalb in verschiedene Tumorassozi ierte Signalwege involviert. Da die Expression der beiden ncRNAs häufig bei Krebs dereguliert ist und einzigartige Signaturen aufweist, spielen sie eine wichtige Rolle in der Pathogenese von Krebs. Sie werden passiv und aktiv in den Blutkreislauf befreit und folglich reflektieren ihre Plasma/Serum-und exosomale Profile die Entwicklung der Krankheit, die Tumorlast und die maligne Progression bis hin zum metastatischen Relaps. Betreffend der Biologie und Natur der ncRNAs in der Regulation der verschiedenen zellulären Prozesse, sind sie als Blut-basierte Marker und zukünftige therapeutische Zielemoleküle bei der Behandlung von Krebspatienten für die Klinik potenziell wichtig. Der vorliegende Reviewartikel befasst sich mit dem diagnostischen Wert von zellfreien und exosomalen ncRNAs im Plasma/Serum von Krebspatienten und den Herausforderungen sie als zukünftige Tumormarker zu etablieren.Schlüsselwörter: Exosomen; flüssige Biopsie; zellfreie miRNAs; zellfreie lncRNAs.

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Data Normalization Strategies for MicroRNA Quantification

Cited by 399 publications

References 90 publications

Direct Comparison of Metastasis-Related miRNAs Expression Levels in Circulating Tumor Cells, Corresponding Plasma, and Primary Tumors of Breast Cancer Patients

Direct Comparison of Metastasis-Related miRNAs Expression Levels in Circulating Tumor Cells, Corresponding Plasma, and Primary Tumors of Breast Cancer Patients

miRNAs as Biomarkers and Therapeutic Targets in Non-Small Cell Lung Cancer: Current Perspectives

Diagnostic relevance of circulating cell-free and exosomal microRNAs and long non-coding RNAs in blood of cancer patients

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