Dataset Information

Analysis of transcription factor mRNAs in identified oxytocin and vasopressin magnocellular neurons isolated by laser capture microdissection.

ABSTRACT: The oxytocin (Oxt) and vasopressin (Avp) magnocellular neurons (MCNs) in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON), and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM), and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV) vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun) are differentially expressed in the Oxt and Avp MCNs.

SUBMITTER: Humerick M

PROVIDER: S-EPMC3722287 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Analysis of transcription factor mRNAs in identified oxytocin and vasopressin magnocellular neurons isolated by laser capture microdissection.

Humerick Madison M Hanson Jeffrey J Rodriguez-Canales Jaime J Lubelski Daniel D Rashid Omar M OM Salinas Yasmmyn D YD Shi Yijun Y Ponzio Todd T Fields Raymond R Emmert-Buck Michael R MR Gainer Harold H

PloS one 20130724 7

The oxytocin (Oxt) and vasopressin (Avp) magnocellular neurons (MCNs) in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON), and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM), and to analyze the differen ...[more]

PMID: 23894472

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Project description:Different cell types and multiple cellular connections characterize the human brain. Gene expression analysis using a specific population of cells is more accurate than conducting analysis of the whole tissue homogenate, particularly in the context of neurodegenerative diseases, where a specific subset of cells is affected by the different pathology. Due to the difficulty of obtaining homogenous cell populations, gene expression in specific cell-types (neurons, astrocytes, etc.) has been understudied. To leverage the use of archive resources of frozen human brains in studies of neurodegenerative diseases, we developed and calibrated a method to quantify cell-type specific-neuronal, astrocytes-expression profiles of genes implicated in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Archive human frozen brain tissues were used to prepare slides for rapid immunostaining using cell-specific antibodies. The immunoreactive-cells were isolated by Laser Capture Microdissection (LCM). The enrichment for a particular cell-type of interest was validated in post-analysis stage by the expression of cell-specific markers. We optimized the technique to preserve the RNA integrity, so that the RNA was suitable for downstream expression analyses. Following RNA extraction, the expression levels were determined digitally using nCounter Single Cell Gene Expression assay (NanoString Technologies®). The results demonstrated that using our optimized technique we successfully isolated single neurons and astrocytes from human frozen brain tissues and obtained RNA of a good quality that was suitable for mRNA expression analysis. We present here new advancements compared to previous reported methods, which improve the method's feasibility and its applicability for a variety of downstream molecular analyses. Our new developed method can be implemented in genetic and functional genomic research of neurodegenerative diseases and has the potential to significantly advance the field.

Dataset Information

Analysis of transcription factor mRNAs in identified oxytocin and vasopressin magnocellular neurons isolated by laser capture microdissection.

Publications

Analysis of transcription factor mRNAs in identified oxytocin and vasopressin magnocellular neurons isolated by laser capture microdissection.

Similar Datasets

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