Potential pitfalls in microRNA profiling

Chugh, Pauline; Dittmer, Dirk P.

doi:10.1002/wrna.1120

Cited by 167 publications

(157 citation statements)

References 114 publications

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“…miRNA precursors have been detected in cell lines and tissues and have been suggested as potential disease markers (Schmittgen et al 2008;O'Hara et al 2009). The quantity of mature miRNA does not necessarily correlate with the quantity of its precursor, due to divergent and unique regulation processes individually influencing each molecule (Chugh and Dittmer 2012).…”

Section: Introductionmentioning

confidence: 99%

“…All these miRNA profiling methods face unique challenges, due to several miRNA characteristics: (1) mature miRNAs are very short; (2) miRNAs share high sequence homology within families, with as low as 1 base difference; (3) miRNAs are known to have a large number of isoforms due to RNA editing, single nucleotide, and length polymorphisms. All the above present obstacles for primer or probe design and hybridization in microarrays or qPCR (for review, see Chugh and Dittmer 2012;Pritchard et al 2012). In NGS, sequence similarity of miRNAs may cause a problem in discriminating between miRNAs due to PCR and sequencing errors.…”

Section: Introductionmentioning

confidence: 99%

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Differences in microRNA detection levels are technology and sequence dependent

Leshkowitz

Horn‐Saban

Parmet

et al. 2013

RNA

106

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Identification and quantification of small RNAs are challenging because of their short length, high sequence similarities within microRNA (miRNA) families, and the existence of miRNA isoforms and O-methyl 3 ′ modifications. In this study, the detection performance of three high-throughput commercial platforms, Agilent and Affymetrix microarrays and Illumina next-generation sequencing, was systematically and comprehensively compared. The ability to detect miRNAs was shown to depend strongly on the platform and on miRNA modifications and sequence. Using synthetic transcripts, including mature, precursor, and Omethyl-modified miRNAs spiked into human RNA, a large intensity variation in all spiked-in miRNAs and a reduced capacity in detecting O-methyl-modified miRNAs were observed between the tested platforms. In addition, endogenous human miRNA expression levels were assessed across the platforms. Detected miRNA expression levels were not consistent between platforms. Although biases in miRNA detection were previously described, here the end-point result, i.e., detection intensity, of these biases was investigated on multiple platforms in a controlled fashion. A detailed exploration of a large number of attributes, including base composition, sequence structure, and isoform miRNA attributes, suggests their impact on miRNA expression detection level. This study provides a basis for understanding the attributes that should be considered to adjust platform-dependent detection biases.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Differences in microRNA detection levels are technology and sequence dependent

Leshkowitz

Horn‐Saban

Parmet

et al. 2013

RNA

106

View full text Add to dashboard Cite

show abstract

“…8,9,11 Moreover, PCR-based systems require great care with primer design due to their target-based amplification, and often introduce sequence bias. 9,12 Microarrays, however, allow large-scale parallel detection of miRNAs, but usually require relatively long assay times with complicated steps. 9,13 Therefore, there is a great demand for systems that provide high sensitivity and specificity without target-based amplification, a simple workflow, and the ability to multiplex small panels of miRNAs in a high throughput manner.…”

mentioning

confidence: 99%

Encoded Hydrogel Microparticles for Sensitive and Multiplex microRNA Detection Directly from Raw Cell Lysates

et al. 2016

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In recent years, microRNAs (miRNAs) have emerged as promising diagnostic markers because of their unique dysregulation patterns under various disease conditions and high stability in biological fluids. However, current methods of analyzing miRNA levels typically require RNA isolation, which is cumbersome and time-consuming. To achieve high-throughput and accurate miRNA profiling, this study eliminates the need for purification steps by detecting miRNA directly from raw cellular lysate using nonfouling polyethylene glycol microparticles. In contrast to recent studies on direct miRNA measurements from cell lysate, our hydrogel-based system provides high-confidence quantification with robust performance. The lysis buffer for the assay was optimized to maximize reaction and labeling efficiency, and this assay has a low limit of detection (<1000 cells) without target amplification. Additionally, the capability for multiplexing was demonstrated through analyzing the levels of three endogenous miRNAs in 3T3 cell lysate. This versatile platform holds great potential for rapid and reliable direct miRNA quantification in complex media, and can be further extended to single-cell analysis by exploiting the flexibility and scalability of our system.

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“…The output delivers sequencing reads of varying lengths corresponding to miRNAs, which are then aligned to the reference sequence of choice. Limitations of this technique include sequence-specific biases due to enzymatic ligation, relatively high cost and the need for trained computer scientists to analyze the abundant data output [18]. A relatively new method uses real-time PCR-based arrays that are available in different sizes (e.g.…”

Section: Techniques Applied For the Quantification Of Ncrnasmentioning

confidence: 99%

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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Potential pitfalls in microRNA profiling

Cited by 167 publications

References 114 publications

Differences in microRNA detection levels are technology and sequence dependent

Differences in microRNA detection levels are technology and sequence dependent

Encoded Hydrogel Microparticles for Sensitive and Multiplex microRNA Detection Directly from Raw Cell Lysates

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

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