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Evaluation of two commercial global miRNA expression profiling platforms for detection of less abundant miRNAs
Steffen G Jensen, Philippe Lamy, Mads H Rasmussen, Marie S Ostenfeld, Lars Dyrskj?t, Torben F ?rntoft, Claus L Andersen
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-435
Abstract: Using synthetic miRNA samples and plasma RNA samples spiked with different ratios of 174 synthetic miRNAs we assessed the performance characteristics reproducibility, recovery, specificity, sensitivity and linearity. It was found that while the qRT-PCR based platforms were sufficiently sensitive to reproducibly detect miRNAs at the abundance levels found in human plasma, the array based platform was not. At high miRNA levels both qRT-PCR based platforms performed well in terms of specificity, reproducibility and recovery. At low miRNA levels, as in plasma, the miRCURY platform showed better sensitivity and linearity than the TaqMan platform.For profiling clinical samples with low miRNA abundance, such as plasma samples, the miRCURY platform with its better sensitivity and linearity would probably be superior.microRNAs (miRNAs) are short 20-23 nucleotide long non-coding RNAs that are widely distributed in almost all eukaryotic organisms. They have multiple functions however the main function is believed to be post transcriptional regulation of protein levels [1,2]. While miRNAs are often abundant in tissues, the amount found circulating in body fluids such as plasma and serum is often limited. It has been reported that the total RNA level in plasma is in the range 6-300 ng/ml [3,4] and that the miRNA fraction constitutes only a few percent of this [5]. The mechanisms regulating secretion of miRNA into circulation is still unclear. Reports have shown that while endogenous miRNAs appear stable in plasma/serum exogenous miRNAs are not, and as a result of this it has been suggested that endogenous circulating miRNAs are either encapsulated in microvesicles or bound to RNA-binding proteins in complexes, e.g. Ago2 and NPM1, protecting them from degradation [6-8]. Detailed knowledge of the biological function of circulating miRNA does not exist, however it has been shown that vesicular miRNAs can be transferred from cell to cell and influence the behavior of the recipient c
MicroRNA Profiling during Cardiomyocyte-Specific Differentiation of Murine Embryonic Stem Cells Based on Two Different miRNA Array Platforms  [PDF]
Lin Gan, Silke Schwengberg, Bernd Denecke
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0025809
Abstract: MicroRNA (miRNA) plays a critical role in a wide variety of biological processes. Profiling miRNA expression during differentiation of embryonic stem cells will help to understand the regulation pathway of differentiation, which in turn may elucidate disease mechanisms. The identified miRNAs could then serve as a new group of possible therapeutic targets. In the present paper, miRNA expression profiles were determined during cardiomyocyte-specific differentiation and maturation of murine embryonic stem (ES) cells. For this purpose a homogeneous cardiomyocyte population was generated from a transgenic murine ES cell line. Two high throughput array platforms (Affymetrix and Febit) were used for miRNA profiling in order to compare the effect of the platforms on miRNA profiling as well as to increase the validity of target miRNA identification. Four time points (i.e. day 0, day 12, day 19 and day 26) were chosen for the miRNA profiling study, which corresponded to different stages during cardiomyocyte-specific differentiation and maturation. Fifty platform and pre-processing method-independent miRNAs were identified as being regulated during the differentiation and maturation processes. The identification of these miRNAs is an important step for characterizing and understanding the events involved in cardiomyocyte-specific differentiation of ES cells and may also highlight candidate target molecules for therapeutic purposes.
Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis
Yongxin Chen, Jonathan AL Gelfond, Linda M McManus, Paula K Shireman
BMC Genomics , 2009, DOI: 10.1186/1471-2164-10-407
Abstract: High reproducibility with qPCR-array was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array.Our studies demonstrated high reproducibility of TaqMan qPCR-array. Comparison between different reverse transcription reactions and qPCR-arrays performed on different days indicated that reverse transcription reactions did not introduce significant variation in the results. The use of cDNA pre-amplification increased the sensitivity of miRNA detection. Although there was variability associated with pre-amplification in low abundance miRNAs, the latter did not involve any systemic bias in the estimation of miRNA expression. Comparison between microarray and qPCR-array indicated superior sensitivity and specificity of qPCR-array.MicroRNAs (miRNAs) are small noncoding RNAs of 20-22 nucleotides in length that direct posttranscriptional regulation through specific recognition of short sequences of target mRNAs, often in the 3' untranslated region, reviewed in [1-3]. With >200 members per species in higher eukaryotes, miRNAs are one of the largest gene families, accounting for ~1% of the genome [4]. Multiple studies have demonstrated that miRNAs are involved in numerous integral biological processes including development, cell proliferation, differentiation and apoptosis, reviewed in [5,6]. The comple
Comparing cDNA and oligonucleotide array data: concordance of gene expression across platforms for the NCI-60 cancer cells
Jae K Lee, Kimberly J Bussey, Fuad G Gwadry, William Reinhold, Gregory Riddick, Sandra L Pelletier, Satoshi Nishizuka, Gergely Szakacs, Jean-Phillipe Annereau, Uma Shankavaram, Samir Lababidi, Lawrence H Smith, Michael M Gottesman, John N Weinstein
Genome Biology , 2003, DOI: 10.1186/gb-2003-4-12-r82
Abstract: Gene expression microarrays are revolutionizing the biomedical sciences, but gross errors in microarray data can arise from a variety of sources, including cross-hybridization, alternative splicing, contamination of clones, mistakes in sequencing, and the fact that hybridization conditions must be 'one-size-fits-all' across an array. Re-sequencing of clones can eliminate some errors in gene identification but not the possibility of a mix-up during the arraying process or the possibility that minor cross-contamination with a clone representing a highly expressed gene will obscure the signal from one of low expression. Therefore, the results for interesting genes are often validated individually by an independent method such as real-time reverse transcription PCR (RT-PCR), northern blot, or RNase protection. With each of these methods, however, the relevant probes or primer-probe sets must be designed, tuned and applied one at a time. Hence, most laboratories can verify the information for only a handful of genes. A multiplexed method that validated thousands of expression levels simultaneously would be preferable.Our strategy for multiplexed validation is to profile a set of RNA samples using two technologies (for example, cDNA microarrays and oligonucleotide chips) that are subject to very different artifacts. When the two technologies disagree, one cannot tell, in the absence of outside information, which is the more accurate. But when they agree, each tends to validate the other. Agreement in a binary experiment (such as cancer versus normal cell type) is better than nothing, but it can be coincidental. Rich patterns of agreement for a given transcript across many samples in a dataset are statistically unlikely to arise by accident. We have found the mutual validation algorithm to be very useful for studies on expression data from the 60 human cancer cell lines (the NCI-60) used by the National Cancer Institute (NCI) to screen for new drug candidates [1,2].The NCI
Quantitative miRNA Expression Analysis Using Fluidigm Microfluidics Dynamic Arrays
Jin Jang, Vernadette A Simon, Rod M Feddersen, Fariborz Rakhshan, Debra A Schultz, Michael A Zschunke, Wilma L Lingle, Christopher P Kolbert, Jin Jen
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-144
Abstract: We demonstrated highly correlated Ct values between multiplex and singleplex RT reactions in standard qPCR assays for miRNA expression using total RNA from A549 (R = 0.98; p < 0.0001) and H1299 (R = 0.95; p < 0.0001) lung cancer cell lines. The Ct values generated by the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left-shift toward lower Ct values compared to those observed by ABI 7900 HT (mean difference, 3.79), suggesting that the microfluidic technology exhibited a greater sensitivity. In addition, we show that as little as 10 ng total RNA can be used to reliably detect all 48 or 96 tested miRNAs using a 96-multiplexing RT reaction in both FFPE and FF samples. Finally, we compared miRNA expression measurements in both FFPE and FF samples by qPCR using the 96.96 dynamic array and Affymetrix microarrays. Fold change comparisons for comparable genes between the two platforms indicated that the overall correlation was R = 0.60. The maximum fold change detected by the Affymetrix microarray was 3.5 compared to 13 by the 96.96 dynamic array.The qPCR-array based microfluidic dynamic array platform can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. We showed that this approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform at a throughput that is 5 to 20 times higher and a sample and reagent usage that was approximately 50-100 times lower than conventional assays. We established optimal conditions for using the Fluidigm microfluidic technology for rapid, cost effective, and customizable arrays for miRNA expression profiling and validation.MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that regulate gene expression by interacting with or inhibiting mRNA in both plants and animals [1-3]. To date, more than 800 human miRNAs have been identified and the total number is still increasing [4]. It is estimated that about two thirds of all
Impact of miRNA Sequence on miRNA Expression and Correlation between miRNA Expression and Cell Cycle Regulation in Breast Cancer Cells  [PDF]
Zijun Luo, Yi Zhao, Robert Azencott
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0095205
Abstract: The miRNAs regulate cell functions by inhibiting expression of proteins. Research on miRNAs had usually focused on identifying targets by base pairing between miRNAs and their targets. Instead of identifying targets, this paper proposed an innovative approach, namely impact significance analysis, to study the correlation between mature sequence, expression across patient samples or time and global function on cell cycle signaling of miRNAs. With three distinct types of data: The Cancer Genome Atlas miRNA expression data for 354 human breast cancer specimens, microarray of 266 miRNAs in mouse Embryonic Stem cells (ESCs), and Reverse Phase Protein Array (RPPA) transfected by 776 miRNAs in MDA-MB-231 cell line, we linked the expression and function of miRNAs by their mature sequence and discovered systematically that the similarity of miRNA expression enhances the similarity of miRNA function, which indicates the miRNA expression can be used as a supplementary factor to predict miRNA function. The results also show that both seed region and 3' portion are associated with miRNA expression levels across human breast cancer specimens and in ESCs; miRNAs with similar seed tend to have similar 3' portion. And we discussed that the impact of 3' portion, including nucleotides , is not significant for miRNA function. These results provide novel insights to understand the correlation between miRNA sequence, expression and function. They can be applied to improve the prediction algorithm and the impact significance analysis can also be implemented to similar analysis for other small RNAs such as siRNAs.
Comprehensive comparison of three commercial human whole-exome capture platforms
Asan, Yu Xu, Hui Jiang, Chris Tyler-Smith, Yali Xue, Tao Jiang, Jiawei Wang, Mingzhi Wu, Xiao Liu, Geng Tian, Jun Wang, Jian Wang, Huangming Yang, Xiuqing Zhang
Genome Biology , 2011, DOI: 10.1186/gb-2011-12-9-r95
Abstract: We comprehensively compared three platforms: NimbleGen's Sequence Capture Array and SeqCap EZ, and Agilent's SureSelect. We assessed their performance in a variety of ways, including number of genes covered and capture efficacy. Differences that may impact on the choice of platform were that Agilent SureSelect covered approximately 1,100 more genes, while NimbleGen provided better flanking sequence capture. Although all three platforms achieved similar capture specificity of targeted regions, the NimbleGen platforms showed better uniformity of coverage and greater genotype sensitivity at 30- to 100-fold sequencing depth. All three platforms showed similar power in exome SNP calling, including medically relevant SNPs. Compared with genotyping and whole-genome sequencing data, the three platforms achieved a similar accuracy of genotype assignment and SNP detection. Importantly, all three platforms showed similar levels of reproducibility, GC bias and reference allele bias.We demonstrate key differences between the three platforms, particularly advantages of solutions over array capture and the importance of a large gene target set.Identifying genetic alterations underlying both rare and common diseases, and also other phenotypic variation, is of particular biological and medical relevance. Even after a decade's effort by the genetics research community since the completion of the first human genome sequences [1,2], most genetic mutations underlying human diseases remain undiscovered. For example, the causative mutations for more than half of human rare diseases [3], the genetic architecture of most common diseases [4,5] and the roles of somatic mutations in most cancers [6] have yet to be characterized. Whole genome re-sequencing can potentially identify these uncharacterized mutations, and in the past few years great strides have been made in this regard with massively parallel DNA sequencing technologies that can be applied to the whole genome [7-10]. However, the c
The miRNA expression profile of the uveal melanoma
ChengHsun Yang,WenBin Wei
Science China Life Sciences , 2011, DOI: 10.1007/s11427-011-4149-y
Abstract: The miRNA expression profile was initially established to investigate its corresponding function in human uveal melanoma. The miRNA expression profile in human uveal melanoma was analyzed by a micro chip technique. The hsa-miRNA expression between four uveal melanomas and four normal uveal tissues was compared. Based on the bioinformatic approach, chip data was analyzed to select out differentially expressed candidate hsa-miRNAs. Real-time quantitative PCR (RT-PCR) was used to confirm the candidate hsa-miRNAs expression in all samples. The results of miRNA microarray chips that matched with RT-PCR were considered as the miRNA expression which was significantly different between normal tissue and uveal melanomas. In four uveal melanomas, expressions of miRNA-20a, miRNA-106a, miRNA-17, miRNA-21, and miRNA-34a were significantly up-regulated, while miRNA-145 and miRNA-204 expression were significantly down-regulated. We used miRNA microarray analysis as a fast, efficient technology to study biological information. The differentially expressed miRNAs may be involved in uveal melanoma pathogenesis, and may help promote the diagnosis and treatment for uveal melanoma.
Systematic Evaluation of Three microRNA Profiling Platforms: Microarray, Beads Array, and Quantitative Real-Time PCR Array  [PDF]
Bin Wang,Paul Howel,Skjalg Bruheim,Jingfang Ju,Laurie B. Owen,Oystein Fodstad,Yaguang Xi
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0017167
Abstract: A number of gene-profiling methodologies have been applied to microRNA research. The diversity of the platforms and analytical methods makes the comparison and integration of cross-platform microRNA profiling data challenging. In this study, we systematically analyze three representative microRNA profiling platforms: Locked Nucleic Acid (LNA) microarray, beads array, and TaqMan quantitative real-time PCR Low Density Array (TLDA).
Impact of Host Genes and Strand Selection on miRNA and miRNA* Expression  [PDF]
Marta Biasiolo, Gabriele Sales, Marta Lionetti, Luca Agnelli, Katia Todoerti, Andrea Bisognin, Alessandro Coppe, Chiara Romualdi, Antonino Neri, Stefania Bortoluzzi
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023854
Abstract: Dysregulation of miRNAs expression plays a critical role in the pathogenesis of genetic, multifactorial disorders and in human cancers. We exploited sequence, genomic and expression information to investigate two main aspects of post-transcriptional regulation in miRNA biogenesis, namely strand selection regulation and expression relationships between intragenic miRNAs and host genes. We considered miRNAs expression profiles, measured in five sizeable microarray datasets, including samples from different normal cell types and tissues, as well as different tumours and disease states. First, the study of expression profiles of “sister” miRNA pairs (miRNA/miRNA*, 5′ and 3′ strands of the same hairpin precursor) showed that the strand selection is highly regulated since it shows tissue-/cell-/condition-specific modulation. We used information about the direction and the strength of the strand selection bias to perform an unsupervised cluster analysis for the sample classification evidencing that is able to distinguish among different tissues, and sometimes between normal and malignant cells. Then, considering a minimum expression threshold, in few miRNA pairs only one mature miRNA is always present in all considered cell types, whereas the majority of pairs were concurrently expressed in some cell types and alternatively in others. In a significant fraction of concurrently expressed pairs, the major and the minor forms found at comparable levels may contribute to post-transcriptional gene silencing, possibly in a coordinate way. In the second part of the study, the behaved tendency to co-expression of intragenic miRNAs and their “host” mRNA genes was confuted by expression profiles examination, suggesting that the expression profile of a given host gene can hardly be a good estimator of co-transcribed miRNA(s) for post-transcriptional regulatory networks inference. Our results point out the regulatory importance of post-transcriptional phases of miRNAs biogenesis, reinforcing the role of such layer of miRNA biogenesis in miRNA-based regulation of cell activities.
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