Antisense RNA Amplification: A Linear Amplification Method for Analyzing the mRNA Population from Single Living Cells

Phillips, Jennifer; Eberwine, James

doi:10.1006/meth.1996.0104

Cited by 220 publications

(127 citation statements)

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“…Transcription of the first strand of cDNA was performed in situ to increase the yield of cDNA from the endogenous mRNA pool before microdissection (Crino et al, 1996;Morrison et al, 2000). After TUNEL and active caspase 3 immunohistochemistry, sections were incubated overnight with the oligo-dT primer coupled with a T7 RNA polymerase promoter sequence (oligo-dT-T7), as described previously (Eberwine et al, 1992b;Phillips and Eberwine, 1996). After firststrand cDNA synthesis, the sections were washed in 0.5ϫ SSC, and individual labeled cells were viewed on an inverted microscope (IX-70; Olympus America , Lake Success, NY) (Crino et al, 1996;O'Dell et al, 2000).…”

Section: Methodsmentioning

confidence: 99%

“…The mRNA from individual neurons was amplified according to previously described methods (Eberwine et al, 1992a,b;Phillips and Eberwine, 1996). Approximately 75% of the double-stranded DNA material was used for first-round aRNA amplification with T7 RNA polymerase (2000 U/l; Epicenter Technologies, Madison, WI), and the remaining 25% was kept for the real-time PCR validation.…”

Section: Methodsmentioning

confidence: 99%

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Neuron-Specific mRNA Complexity Responses during Hippocampal Apoptosis after Traumatic Brain Injury

Marciano¹,

Brettschneider²,

Manduchi³

et al. 2004

J. Neurosci.

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In an effort to understand the complexity of genomic responses within selectively vulnerable regions after experimental brain injury, we examined whether single apoptotic neurons from both the CA3 and dentate differed from those in an uninjured brain. The mRNA from individual active caspase 3(ϩ)/terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling [TUNEL(Ϫ)] and active caspase 3(ϩ)/TUNEL(ϩ) pyramidal and granule neurons in brain-injured mice were amplified and compared with those from nonlabeled neurons in uninjured brains. Gene analysis revealed that overall expression of mRNAs increased with activation of caspase 3 and decreased to below uninjured levels with TUNEL reactivity. Cell type specificity of the apoptotic response was observed with both regionally distinct expression of mRNAs and differences in those mRNAs that were maximally regulated. Immunohistochemical analysis for two of the most highly differentially expressed genes ( prion and Sos2) demonstrated a correlation between the observed differential gene expression after traumatic brain injury and corresponding protein translation.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Neuron-Specific mRNA Complexity Responses during Hippocampal Apoptosis after Traumatic Brain Injury

Marciano¹,

Brettschneider²,

Manduchi³

et al. 2004

J. Neurosci.

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“…The principles of this amplification have been described previously (Phillips and Eberwine, 1996). The first round of amplification was performed on 150 ng of the pooled hypothalamic reference and 150 ng of laser-microdissected SON material.…”

Section: T7-based Rna Amplificationmentioning

confidence: 99%

Selective Gene Expression in Magnocellular Neurons in Rat Supraoptic Nucleus

Mutsuga¹,

Shahar²,

Verbalis³

et al. 2004

J. Neurosci.

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Oxytocin-and vasopressin-producing magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system are the only neuronal phenotypes present in the rat supraoptic nucleus (SON). Laser microdissection of the SON, extraction and T7-based amplification of its RNAs, and analysis of the resulting cDNAs by hybridization on a 35, 319 element DNA microarray have provided a detailed composite view of the gene expression profile of the MCNs. The genes expressed in the SON were compared with those expressed in a reference tissue consisting of total hypothalamus, and this "expression ratio" indicated which genes were preferentially expressed in the SON. Of the 26,000 unique genes on the array, 1385 were found to be expressed in the SON at levels more than two times greater than in the hypothalamus as a whole. Of these, 123 were expressed Ն3.4-fold higher in the SON versus hypothalamus. Most of these preferentially expressed genes were not previously known to be expressed in the MCNs. Quantitative and double-label in situ hybridization histochemistry was used selectively to confirm a number of these microarray observations and to evaluate the osmotic regulation and cell-specific expression of these genes, respectively.

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“…In this strategy, polyA RNA is primed for cDNA synthesis by a polyT oligonucleotide containing the 17 bp sequence for the T7 RNA polymerase promoter. After second-strand synthesis is completed, the template is then transcribed by a highly concentrated T7 polymerase, resulting in a B2000-fold amplification of antisense RNA that can be used for hybridisation analysis (Phillips and Eberwine, 1996).…”

Section: Mrna Amplificationmentioning

confidence: 99%