Potential pitfalls in microRNA profiling.

Department of Microbiology, UNC-Chapel Hill, Chapel Hill, NC, USA.
WIREs RNA (Impact Factor: 6.15). 05/2012; 3(5):601-16. DOI: 10.1002/wrna.1120
Source: PubMed

ABSTRACT MicroRNAs (miRNAs) are small, noncoding RNAs that post-transcriptionally influence a wide range of cellular processes such as the host response to viral infection, innate immunity, cell cycle progression, migration, and apoptosis through the inhibition of target mRNA translation. Owing to the growing number of miRNAs and identification of their functional roles, miRNA profiling of many different sample types has become more expansive, especially with relevance to disease signatures. In this review, we address some of the advantages and potential pitfalls of the currently available methods for miRNA expression profiling. Some of the topics discussed include isomiRNAs, comparison of different profiling platforms, normalization strategies, and issues with regard to sample preparation and experimental analyses.

1 Bookmark
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The innate immune response to West Nile virus (WNV) infection involves recognition through toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), leading to establishment of an antiviral state. MiRNAs (miRNAs) have been shown to be reliable biomarkers of TLR activation. Here, we sought to evaluate the contribution of TLR3 and miRNAs to the host response to WNV infection. We first analyzed HEK293-NULL and HEK293-TLR3 cells for changes in the innate immune response to infection. The presence of TLR3 did not seem to affect WNV load, infectivity or phosphorylation of IRF3. Analysis of experimentally validated NFκB-responsive genes revealed a WNV-induced signature largely independent of TLR3. Since miRNAs are involved in viral pathogenesis and the innate response to infection, we sought to identify changes in miRNA expression upon infection in the presence or absence of TLR3. MiRNA profiling revealed 70 miRNAs induced following WNV infection in a TLR3-independent manner. Further analysis of predicted gene targets of WNV signature miRNAs revealed genes highly associated with pathways regulating cell death, viral pathogenesis and immune cell trafficking.
    PLoS ONE 08/2014; 9(8):e104770. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: This report describes technologies to identify and quantify microRNAs (miRNAs) as potential cancer biomarkers, using breast cancer as an example. Most breast cancer patients are not diagnosed until the disease has advanced to later stages, which decreases overall survival rates. Specific miRNAs are up- or downregulated in breast cancer patients at various stages, can be detected in plasma and serum, and have shown promising preliminary clinical sensitivity and specificity for early cancer diagnosis or staging. Nucleic acid testing methods to determine relative concentrations of selected miRNAs include reverse transcription, followed by quantitative PCR (RT-qPCR), microarrays, and next-generation sequencing (NGS). Of these methods, NGS is the most powerful approach for miRNA biomarker discovery, whereas RT-qPCR shows the most promise for eventual clinical diagnostic applications. © 2014 Society for Laboratory Automation and Screening.
    Journal of the Association for Laboratory Automation 12/2014; · 1.50 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: MicroRNAs (miRNAs) are ∼20-24 nucleotide-long regulatory RNAs that have been proven to play important roles in many cellular processes. Since their discovery, a number of different techniques have been developed to detect and accurately quantify them. For individual mature miRNA measurements, quantitative stem-loop real-time PCR represents a widely used method. Although there are some data on optimization of this technique, there are still many factors that have not been investigated yet. In this study, we have thoroughly optimized this technique and pointed out several important factors that influence reliable quantification. First, we found that total RNA input can affect the measurements. Second, our data showed that carryover DNA contamination could also mislead the detection in a sequence-specific manner. Additionally, we provided evidence that different 3' isomiR species of a particular miRNA can be reverse transcribed and cross-detected even by specifically targeted assays. Besides these, we have investigated the measurement of reaction efficiencies from total RNA samples and the accuracy of simultaneous reverse transcription reactions for increasing reliability and cost effectiveness without the loss of sensitivity and specificity. In summary, we provide a detailed, refined protocol for reliable detection of microRNA species by quantitative stem-loop PCR.
    PLoS ONE 08/2014; 9(8):e106315. · 3.53 Impact Factor

Full-text (2 Sources)

Available from
May 28, 2014