Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

Carter, Mark G.; Sharov, Alexei A.; VanBuren, Vincent; Dudekula, Dawood B.; Carmack, Condie E.; Nelson, Charlie; Ko, Minoru S.H.

doi:10.1186/gb-2005-6-7-r61

Cited by 113 publications

(54 citation statements)

References 31 publications

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“…For comparison, we used ESCs in standard culture conditions as well as cells derived from secondary colonies and then cultured 10 passages in standard conditions. Cy3-CTP-labeled sample targets (in two biological replications) were prepared with total RNA by Low RNA Input Fluorescent Linear Amplification Kit (Agilent, Santa Clara, CA) and hybridized to NIA Mouse 44K Microarray v3.0 (Agilent, design ID 015087) (Carter et al 2005) together with Cy5-CTP labeled reference target, which was produced from mixture of Stratagene Universal Mouse Reference RNA and RNA from MC1 cells. For statistical analysis, we used NIA Array Analysis, which estimates the False Discovery Rate (FDR) to account for multiple hypothesis testing (Sharov et al 2005).…”

Section: Methodsmentioning

confidence: 99%

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment

Sharova

Sharov

Piao

et al. 2016

In Vitro Cell.Dev.Biol.-Animal

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Retinoic acid (RA) is one of the most potent inducers of differentiation of mouse embryonic stem cells (ESCs). However, previous studies show that RA treatment of cells cultured in the presence of a leukemia inhibitory factor (LIF) also result in the upregulation of a gene called Zscan4, whose transient expression is a marker for undifferentiated ESCs. We explored the balance between these two seemingly antagonistic effects of RA. ESCs indeed differentiated in the presence of LIF after RA treatment, but colonies of undifferentiated ESCs eventually emerged from these differentiated cells — even in the presence of RA. These colonies, named secondary colonies, consist of three cell types: typical undifferentiated ESCs expressing pluripotency genes such as Pou5f1, Sox2, and Nanog; cells expressing Zscan4; and endodermal-like cells located at the periphery of the colony. The capacity to form secondary colonies was confirmed for all eight tested ESC lines. Cells from the secondary colonies — after transfer to the standard ESC medium — retained pluripotency, judged by their strong alkaline phosphatase (ALP) staining, typical colony morphology, gene expression profile, stable karyotype, capacity to differentiate into all three germ layers in embryoid body formation assays, and successful contribution to chimeras after injection into blastocysts. Based on flow cytometry analysis (FACS), the proportion of Zscan4-positive cells in secondary colonies was higher than in standard ESC colonies, which may explain the capacity of ESCs to resist the differentiating effects of RA and instead form secondary colonies of undifferentiated ESCs. This hypothesis is supported by cell-lineage tracing analysis, which showed that most cells in the secondary colonies were descendents of cells transiently expressing Zscan4.

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Section: Methodsmentioning

confidence: 99%

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment

Sharova

Sharov

Piao

et al. 2016

In Vitro Cell.Dev.Biol.-Animal

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“…We performed quantitative RT-PCR using our precapture RNA sample to provide an informed estimate of lncRNA transcript copy number. Assuming an average human fibroblast cell contains ~300 fg of mRNA per cell 27 , we estimate that the lncRNAs we discovered were present at an average of ~0.0006 transcripts per cell, indicating expression in only a small subpopulation of the cells sampled. By comparison, we calculate HOXA to be present at an average ~0.13 transcripts per cell, consistent with previous estimates 27 .…”

mentioning

confidence: 90%

Targeted RNA sequencing reveals the deep complexity of the human transcriptome

et al. 2011

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Transcriptomic analyses have revealed an unexpected complexity to the human transcriptome, whose breadth and depth exceeds current RNA sequencing capability1–4. Using tiling arrays to target and sequence select portions of the transcriptome, we identify and characterize unannotated transcripts whose rare or transient expression is below the detection limits of conventional sequencing approaches. We use the unprecedented depth of coverage afforded by this technique to reach the deepest limits of the human transcriptome, exposing widespread, regulated and remarkably complex noncoding transcription in intergenic regions, as well as unannotated exons and splicing patterns in even intensively studied protein-coding loci such as p53 and HOX. The data also show that intermittent sequenced reads observed in conventional RNA sequencing data sets, previously dismissed as noise, are in fact indicative of unassembled rare transcripts. Collectively, these results reveal the range, depth and complexity of a human transcriptome that is far from fully characterized.

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“…Therefore, although the different available microarray platforms for mRNA expression profiling have been extensively evaluated (Canales et al 2006), there is still no gold standard for quantifying mRNA transcript concentrations by microarrays. The approaches developed so far encompass mathematical modeling (Hekstra et al 2003;Frigessi et al 2005), calibrated reference samples (Dudley et al 2002), exogenous RNA controls (Carter et al 2005b), and combinations of mathematical modeling and spike measurements (Zhao et al 2007). The TransCount method (Frigessi et al 2005) is based on Bayesian statistical modeling and utilizes covariants of the microarray experiment to calculate the concentration from the signal intensity of each probe.…”

Section: Discussionmentioning

confidence: 99%

Absolute quantification of microRNAs by using a universal reference

Bissels¹,

Wild²,

Tomiuk³

et al. 2009

RNA

181

132

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MicroRNAs (miRNAs) are a species of small RNAs ;21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less useful as an assay for absolute quantification. Here, we present a microarray-based approach for global and absolute quantification of miRNAs. The method relies on the parallel hybridization of the sample of interest labeled with Cy5 and a universal reference of 954 synthetic miRNAs in equimolar concentrations that are labeled with Cy3 on a microarray slide containing probes for all human, mouse, rat, and viral miRNAs (miRBase 12.0). Each single miRNA is quantified with respect to the universal reference canceling biases related to sequence, labeling, or hybridization. We demonstrate the accuracy of the method by various spike-in experiments. Furthermore, we quantified miRNA copy numbers in liver samples and CD34(+)/ CD133(À) hematopoietic progenitor cells.

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Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

Cited by 113 publications

References 31 publications

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment

Emergence of undifferentiated colonies from mouse embryonic stem cells undergoing differentiation by retinoic acid treatment

Targeted RNA sequencing reveals the deep complexity of the human transcriptome

Absolute quantification of microRNAs by using a universal reference

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