Systematic evaluation of medium-throughput mRNA abundance platforms

Prokopec, Stephenie D.; Watson, John D.; Waggott, Daryl; Smith, Ashley B.; Wu, Alexander H.; Okey, Allan B.; Pohjanvirta, Raimo; Boutros, Paul C.

doi:10.1261/rna.034710.112

Cited by 80 publications

(73 citation statements)

References 44 publications

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“…validated previously (Pohjanvirta et al, 2006) for use in TCDD studies of rat hypothalamus. Similarly, this normalization method had been previously identified as the most accurate (in comparison to qPCR) for use in a similar study with rat hepatic tissue (Prokopec et al, 2013). Normalized data was log 2 -transformed and linear modelling and visualizations performed as above [R packages: limma (v3.20.9), lattice (v0.20-29), latticeExtra (v0.6-26)].…”

Section: Validationmentioning

confidence: 99%

Transcriptional profiling of rat hypothalamus response to 2,3,7,8-tetrachlorodibenzo-ρ-dioxin

et al. 2015

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In some mammals, halogenated aromatic hydrocarbon (HAH) exposure causes wasting syndrome, defined as significant weight loss associated with lethal outcomes. The most potent HAH in causing wasting is 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), which exerts its toxic effects through the aryl hydrocarbon receptor (AHR). Since TCDD toxicity is thought to predominantly arise from dysregulation of AHR-transcribed genes, it was hypothesized that wasting syndrome is a result of to TCDD-induced dysregulation of genes involved in regulation of food-intake. As the hypothalamus is the central nervous systems' regulatory center for food-intake and energy balance. Therefore, mRNA abundances in hypothalamic tissue from two rat strains with widely differing sensitivities to TCDD-induced wasting syndrome: TCDD-sensitive Long-Evans rats and TCDD-resistant Han/Wistar rats, 23h after exposure to TCDD (100μg/kg) or corn oil vehicle. TCDD exposure caused minimal transcriptional dysregulation in the hypothalamus, with only 6 genes significantly altered in Long-Evans rats and 15 genes in Han/Wistar rats. Two of the most dysregulated genes were Cyp1a1 and Nqo1, which are induced by TCDD across a wide range of tissues and are considered sensitive markers of TCDD exposure. The minimal response of the hypothalamic transcriptome to a lethal dose of TCDD at an early time-point suggests that the hypothalamus is not the predominant site of initial events leading to hypophagia and associated wasting. TCDD may affect feeding behaviour via events upstream or downstream of the hypothalamus, and further work is required to evaluate this at the level of individual hypothalamic nuclei and subregions.

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Section: Validationmentioning

confidence: 99%

Transcriptional profiling of rat hypothalamus response to 2,3,7,8-tetrachlorodibenzo-ρ-dioxin

et al. 2015

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“…In essence, this calculation accounts for different amounts of total number of counted molecules across samples (45). Data were log-normalized (base 2), and all microRNAs with maximum normalized expression Ͻ 6.0 were removed from subsequent analysis.…”

Section: Methodsmentioning

confidence: 99%

MicroRNA network changes in the brain stem underlie the development of hypertension

DeCicco

Zhu

Brureau

et al. 2015

Physiological Genomics

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sion is a major chronic disease whose molecular mechanisms remain poorly understood. We compared neuroanatomical patterns of microRNAs in the brain stem of the spontaneous hypertensive rat (SHR) to the Wistar Kyoto rat (WKY, control). We quantified 419 wellannotated microRNAs in the nucleus of the solitary tract (NTS) and rostral ventrolateral medulla (RVLM), from SHR and WKY rats, during three main stages of hypertension development. Changes in microRNA expression were stage-and region-dependent, with a majority of SHR vs. WKY differential expression occurring at the hypertension onset stage in NTS versus at the prehypertension stage in RVLM. Our analysis identified 24 microRNAs showing time-dependent differential expression in SHR compared with WKY in at least one brain region. We predicted potential gene regulatory targets corresponding to catecholaminergic processes, neuroinflammation, and neuromodulation using the miRWALK and RNA22 databases, and we tested those bioinformatics predictions using high-throughput quantitative PCR to evaluate correlations of differential expression between the microRNAs and their predicted gene targets. We found a novel regulatory network motif consisting of microRNAs likely downregulating a negative regulator of prohypertensive processes such as angiotensin II signaling and leukotriene-based inflammation. Our results provide new evidence on the dynamics of microRNA expression in the development of hypertension and predictions of microRNA-mediated regulatory networks playing a region-dependent role in potentially altering brain-stem cardiovascular control circuit function leading to the development of hypertension.hypertension; microRNA; neuroinflammation; angiotensin II; brain stem HYPERTENSION IS A MAJOR CHRONIC disease worldwide, affecting about 50 million adults in the United States alone. It is a multiorgan disease and a major cause of heart failure, coronary heart disease, atrial fibrillation, stroke, and end-stage renal disease, and it has an enormous economic impact. About 90 -95% of cases are "essential" with no known medical cause (11). Moreover, only about a third of hypertensive patients have their blood pressure fully controlled (11). A significant and increasing population (ϳ25%) of hypertensive patients is drug resistant. There remains an unmet need for antihypertensive therapy.In the context of the recent discovery of novel lines of therapeutic interventions involving approaches to modulate microRNA we decided to elucidate changes in microRNA levels during the development of spontaneous hypertensive rat (SHR) hypertension. The recent explosion in microRNA research has produced new computational and experimental approaches for studying microRNA functions in cell culture and in vivo, which we take advantage of here. MicroRNAs are an abundant class of small (ϳ22 nt) endogenous noncoding RNAs that direct posttranscriptional regulation of gene expression. There is ample evidence that dysregulation of microRNAs is associated with the pathogenesis of human diseases, in...

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“…These types of studies require a rapid, simple method to assess oligonucleotide-dependent target gene silencing in vivo. The most common method to quantify target mRNA silencing following siRNA treatment is quantitative real-time polymerase chain reaction (qRT-PCR) [9,10].…”

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confidence: 99%

“…qRT-PCR is one of the most widely used methods of analyzing gene expression, and it is an indispensable tool for mRNA quantification for a variety of applications [10][11][12][13]. This assay monitors the exponential amplification of a target mRNA following conversion to its complementary DNA (cDNA).…”

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confidence: 99%

“…However, each step (mRNA isolation and purification, cDNA synthesis, and qRT-PCR) introduces systematic error [9]. These errors include insufficient or nonreproducible mRNA purification from tissues of interest, inefficient cDNA synthesis, and errors in detection of PCR products [10]. Moreover, the purified RNA is subject to degradation by endoand exonucleases [14].…”

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confidence: 99%

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A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

Coles

Osborn

Alterman

et al. 2016

Nucleic Acid Therapeutics

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Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene Ò branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra-and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo.

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Systematic evaluation of medium-throughput mRNA abundance platforms

Cited by 80 publications

References 44 publications

Transcriptional profiling of rat hypothalamus response to 2,3,7,8-tetrachlorodibenzo-ρ-dioxin

Transcriptional profiling of rat hypothalamus response to 2,3,7,8-tetrachlorodibenzo-ρ-dioxin

MicroRNA network changes in the brain stem underlie the development of hypertension

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

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