Quantitative analysis of mRNA amplification by in vitro transcription

Baugh, L. Ryan; Hill, Andrew; Brown, Eugene L.; Hunter, Craig P.

doi:10.1093/nar/29.5.e29

Cited by 455 publications

(404 citation statements)

References 27 publications

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“…The small quantities of material obtained from either cells or dissected cochlear tissues necessitate a twofold approach: the amplification of total RNA (Van Gelder et al 1990;Baugh et al 2001;Xiang et al 2003) and a signal amplification of fluorescence signals using dendrimer technology (Stears et al 2000). Both these strategies were incorporated in our studies.…”

Section: Discussionmentioning

confidence: 99%

“…Amplified RNA (aRNA) was synthesized using one round of a T7-based linear amplification protocol as described (Van Gelder et al 1990;Baugh et al 2001 gen). The reaction was incubated at 16-C for 2 h. Afterwards, the cDNA was purified and concentrated using the DNAclear kit (Ambion).…”

Section: Rna Amplificationmentioning

confidence: 99%

“…This requirement has mandated the use of whole inner ear/cochlear preparations, which can effectively Bdilute out[ the identification and quantification of pertinent transcripts. Recent technical advances in amplification of transcripts (Baugh et al 2001;Badiee et al 2003) and probe signal (Capaldi et al 2000) have drastically reduced the required amount of starting material.…”

Section: Introductionmentioning

confidence: 99%

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Differential Expression of Genes within the Cochlea as Defined by a Custom Mouse Inner Ear Microarray

Morris

Snir²,

Pompéia

et al. 2005

JARO

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Microarray analyses have contributed greatly to the rapid understanding of functional genomics through the identification of gene networks as well as gene discovery. To facilitate functional genomics of the inner ear, we have developed a mouse inner-earpertinent custom microarray chip (CMA-IE1). Nonredundant cDNA clones were obtained from two cDNA library resources: the RIKEN subtracted inner ear set and the NIH organ of Corti library. At least 2000 cDNAs unique to the inner ear were present on the chip. Comparisons were performed to examine the relative expression levels of these unique cDNAs within the organ of Corti, lateral wall, and spiral ganglion. Total RNA samples were obtained from the three cochlear-dissected fractions from adult CF-1 mice. The total RNA was linearly amplified, and a dendrimer-based system was utilized to enhance the hybridization signal. Differentially expressed genes were verified by comparison to known gene expression patterns in the cochlea or by correlation with genes and gene families deduced to be present in the three tissue types. Approximately 22Y25% of the genes on the array had significant levels of expression. A number of differentially expressed genes were detected in each tissue fraction. These included genes with known functional roles, hypothetical genes, and various unknown or uncharacterized genes. Four of the differentially expressed genes found in the organ of Corti are linked to deafness loci. None of these are hypothetical or unknown genes.

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Section: Discussionmentioning

confidence: 99%

Section: Rna Amplificationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Differential Expression of Genes within the Cochlea as Defined by a Custom Mouse Inner Ear Microarray

Morris

Snir²,

Pompéia

et al. 2005

JARO

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“…RNA concentration was determined using a NanoDrop spectrophotometer from NanoDrop Technologies (Rockland, DE), which allows accurate measurement of nanogram amounts of RNA. FACS-purified astrocytes yielded 130-350 ng total RNA, and a 30 ng aliquot of the total RNA was carried through a two-round amplification (Baugh et al, 2001;Sohn et al, 2006). In brief, double-stranded cDNA was synthesized from 30 ng of total RNA with an oligdT primer containing the T7 RNA polymerase promoter (Geneset, LaJolla, CA) and SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA).…”

Section: Rna Isolation and Amplification And Cdna Synthesismentioning

confidence: 99%

Gene expression profiling of astrocytes from hyperammonemic mice reveals altered pathways for water and potassium homeostasis in vivo

Lichter‐Konecki

Mangin

Gordish‐Dressman

et al. 2008

Glia

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Acute hyperammonemia (HA) causes cerebral edema and brain damage in children with urea cycle disorders (UCDs) and in patients in acute liver failure. Chronic HA is associated with developmental delay and mental retardation in children with UCDs, and with neuropsychiatric symptoms in patients with chronic liver failure. Astrocytes are a major cellular target of hyperammonemic encephalopathy, and changes occurring in these cells are thought to be causally related to the brain edema of acute HA. To study the effect of HA on astrocytes in vivo, we crossed the Otc spf mouse, a mouse with the X-linked UCD ornithine transcarbamylase (OTC) deficiency, with the hGFAP-EGFP mouse, a mouse selectively expressing green fluorescent protein in astrocytes. We used FACS to purify astrocytes from the brains of hyperammonemic and healthy Otc spf /GFAP-EGFP mice. RNA isolated from these astrocytes was used in microarray expression analyses and qRT-PCR. When compared with healthy littermates, we observed a significant downregulation of the gap-junction channel connexin 43 (Cx43) the water channel aquaporin 4 (Aqp4) genes, and the astrocytic inward-rectifying potassium channel (Kir) genes Kir4.1 and Kir5.1 in hyperammonemic mice. Aqp4, Cx43, and Kir4.1/ Kir5.1 are co-localized to astrocytic end-feet at the brain vasculature, where they regulate potassium and water transport.Since, ions can permeate water and K + -channels, downregulation of these three channels may be a direct effect of elevated blood ammonia levels. Our results suggest that alterations in astrocyte-mediated water and potassium homeostasis in brain may be key to the development of the brain edema.

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“…27,28 cDNA samples were purified by phenol/ chloroform/IAA/PLG extraction, precipitated and used to generate biotinylated cRNA by in vitro transcription for 16 hr at 37°C (Bioarray High Yield RNA Transcript Labeling Kit; Enzo Life Sciences, Farmingdale, NY). Purification of cRNA was done using RNeasy mini columns (Qiagen).…”

Section: Expression Analysismentioning

confidence: 99%

Genomic gains on chromosome 1q in retinoblastoma: Consequences on gene expression and association with clinical manifestation

Gratias

Schüler

Hitpass

et al. 2005

Intl Journal of Cancer

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Many retinoblastomas (Rbs) show genomic alterations in addition to mutational loss of both normal RB1 alleles. The most frequent of these changes are gains on chromosomes 1q and 6p and losses on 16q. To identify the genes targeted by gains on chromosome 1q, we used quantitative-multiplex PCR to determine DNA copy number changes in 76 primary tumors and 6 Rb cell lines. In addition, in 21 of these tumors, gene expression was analyzed by cDNA microarray hybridization. Increased copy numbers of loci on chromosome 1q were present in 34 (45%) primary tumors and in all 6 cell lines. Two regions of gain emerged, one in 1q32 and another in 1q21. Tumors with 1q gains showed higher RNA expression of several genes in these 2 regions. The clinical manifestation of tumors with and without gains was similar with regard to many aspects, including size, necrosis and calcification. However, the distribution of age at diagnosis was remarkably distinct, with earlier diagnosis in tumors without gains. This suggests that these tumors either are initiated earlier or grow faster than tumors with gains. This association with clinical manifestation indicates that gains on 1q are significant for the biology of Rb. The genes on 1q with copy number gains and overexpression are candidates that need to be tested for their individual contribution to the progression of Rb. ' 2005 Wiley-Liss, Inc.

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Quantitative analysis of mRNA amplification by in vitro transcription

Cited by 455 publications

References 27 publications

Differential Expression of Genes within the Cochlea as Defined by a Custom Mouse Inner Ear Microarray

Differential Expression of Genes within the Cochlea as Defined by a Custom Mouse Inner Ear Microarray

Gene expression profiling of astrocytes from hyperammonemic mice reveals altered pathways for water and potassium homeostasis in vivo

Genomic gains on chromosome 1q in retinoblastoma: Consequences on gene expression and association with clinical manifestation

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