NanoStriDE: normalization and differential expression analysis of NanoString nCounter data

Brumbaugh, Christopher Drew; Kim, Hyunsung John; Giovacchini, Mario; Pourmand, Nader

doi:10.1186/1471-2105-12-479

Cited by 57 publications

(48 citation statements)

References 10 publications

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“…A total of 100 ng of total RNA was assayed using the Human nCounter miRNA Assay 2.0 Kit following the manufacturer’s instructions (NanoString). Differences in miRNA expression were analyzed using the NanoSTRIDE software program (18) with default settings. Clustering of the differentially expressed genes and heatmap generation was performed using the GenePattern Server (genepattern.broadinstitute.org).…”

Section: Methodsmentioning

confidence: 99%

Loss of Estrogen-Regulated microRNA Expression Increases HER2 Signaling and Is Prognostic of Poor Outcome in Luminal Breast Cancer

2015

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Among the genes regulated by estrogen receptor (ER) are miRNAs that play a role in breast cancer signaling pathways. To determine whether miRNAs are involved in ER-positive breast cancer progression to hormone independence, we profiled the expression of 800 miRNAs in the estrogen-dependent human breast cancer cell line MCF7 and its estrogen-independent derivative MCF7:2A (MCF7:2A) using NanoString. We found 78 miRNAs differentially expressed between the two cell lines, including a cluster comprising let-7c, miR-99a, and miR-125b, which is encoded in an intron of the long non-coding RNA LINC00478. These miRNAs are ER targets in MCF7 cells, and nearby ER binding and their expression is significantly decreased in MCF7:2A cells. The expression of these miRNAs was interrogated in patient samples profiled in The Cancer Genome Atlas (TCGA). Among luminal tumors, these miRNAs are expressed at higher levels in luminal A vs. B tumors. While their expression is uniformly low in luminal B tumors, they are lost only in a subset of luminal A patients. Interestingly, this subset with low expression of these miRNAs had worse overall survival compared with luminal A patients with high expression. We confirmed that miR-125b directly targets HER2 and that let-7c also regulates HER2 protein expression. In addition, HER2 protein expression and activity is negatively correlated with let-7c expression in TCGA. In summary, we identified an ER-regulated miRNA cluster that regulates HER2, is lost with progression to estrogen independence, and may serve as a biomarker of poor outcome in ER+ luminal A breast cancer patients.

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Section: Methodsmentioning

confidence: 99%

Loss of Estrogen-Regulated microRNA Expression Increases HER2 Signaling and Is Prognostic of Poor Outcome in Luminal Breast Cancer

2015

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“…Briefly, input RNA (105 ng) and miRtag linkers were ligated prior to hybridization with barcoded reporter and biotinylated capture probes at 65°C for 16 h. Samples were prepared for analysis on the nCounter Prep Station before data were collected at 555 FOV on the nCounter Digital Analyzer. Data were analyzed using the NanoString Differential Expression (NanoStriDE) interface (Brumbaugh et al 2011). Genome-wide miRNA abundance was normalized using a set of housekeeping mRNAs (Supplemental Table S10).…”

Section: Quantification Of Mirna Abundancementioning

confidence: 99%

Extensive microRNA-mediated crosstalk between lncRNAs and mRNAs in mouse embryonic stem cells

et al. 2015

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Recently, a handful of intergenic long noncoding RNAs (lncRNAs) have been shown to compete with mRNAs for binding to miRNAs and to contribute to development and disease. Beyond these reports, little is yet known of the extent and functional consequences of miRNA-mediated regulation of mRNA levels by lncRNAs. To gain further insight into lncRNA-mRNA miRNA-mediated crosstalk, we reanalyzed transcriptome-wide changes induced by the targeted knockdown of over 100 lncRNA transcripts in mouse embryonic stem cells (mESCs). We predicted that, on average, almost one-fifth of the transcript level changes induced by lncRNAs are dependent on miRNAs that are highly abundant in mESCs. We validated these findings experimentally by temporally profiling transcriptome-wide changes in gene expression following the loss of miRNA biogenesis in mESCs. Following the depletion of miRNAs, we found that >50% of lncRNAs and their miRNA-dependent mRNA targets were up-regulated coordinately, consistent with their interaction being miRNA-mediated. These lncRNAs are preferentially located in the cytoplasm, and the response elements for miRNAs they share with their targets have been preserved in mammals by purifying selection. Lastly, miRNA-dependent mRNA targets of each lncRNA tended to share common biological functions. Post-transcriptional miRNA-mediated crosstalk between lncRNAs and mRNA, in mESCs, is thus surprisingly prevalent, conserved in mammals, and likely to contribute to critical developmental processes.

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“…We use genes thereafter for convenience. Most current methods for DE detection in nCounter data, such as NanoStringNorm (3) and NanoStriDE (4), are based on t-tests. Although popular, the t-test is most appropriate for analyzing continuous, preferably normally distributed data.…”

Section: Introductionmentioning

confidence: 99%

Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method

Wang¹,

Beal²

2016

Nucleic Acids Research

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Adenosine deamination is one of the most prevalent post-transcriptional modifications in mRNA. In humans, ADAR1 and ADAR2 catalyze this modification and their malfunction correlates with disease. Recently our laboratory reported crystal structures of the human ADAR2 deaminase domain bound to duplex RNA revealing a protein loop that binds the RNA on the 5′ side of the modification site. This 5′ binding loop appears to be one contributor to substrate specificity differences between ADAR family members. In this study, we endeavored to reveal detailed structure–activity relationships in this loop to advance our understanding of RNA recognition by ADAR2. To achieve this goal, we established a high-throughput mutagenesis approach which allows rapid screening of ADAR variants in single yeast cells and provides quantitative evaluation for enzymatic activity. Using this approach, we determined the importance of specific amino acids at 19 different positions in the ADAR2 5′ binding loop and revealed six residues that provide essential structural elements supporting the fold of the loop and key RNA-binding functional groups. This work provided new insight into RNA recognition by ADAR2 and established a new tool for defining structure–function relationships in ADAR reactions.

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NanoStriDE: normalization and differential expression analysis of NanoString nCounter data

Cited by 57 publications

References 10 publications

Loss of Estrogen-Regulated microRNA Expression Increases HER2 Signaling and Is Prognostic of Poor Outcome in Luminal Breast Cancer

Loss of Estrogen-Regulated microRNA Expression Increases HER2 Signaling and Is Prognostic of Poor Outcome in Luminal Breast Cancer

Extensive microRNA-mediated crosstalk between lncRNAs and mRNAs in mouse embryonic stem cells

Probing RNA recognition by human ADAR2 using a high-throughput mutagenesis method

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