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Immuno-laser capture microdissection of perfusion-fixed brain tissue coupled to RNA-seq

ABSTRACT: Immuno-laser capture microdissection of perfusion-fixed brain tissue coupled to RNA-seq

PROVIDER: PRJNA1259776 | ENA |

REPOSITORIES: ENA

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Immuno-laser capture microdissection of perfusion-fixed brain tissue coupled to RNA-seq

Project description:Normal mouse brain was fixed by cardiac perfusion with 4% paraformaldehyde in PBS, then post-fixed by immersion in the same solution for 2hr at RT. Brain was embedded in cryomatrix and frozen sections were cut at 7 um on a cryostat. Sections were immunostained with anti-CD31 antibody and anti-GFAP antibody to detect microvascular endothelial cells and surrounding astrocytes, respectively. Immediately after immunostaining, sections were subject to laser capture microdissection (LCM), using ultraviolet laser to ablate unwanted astrocytes and infrared laser to lift target endothelial tissue. Total RNA was isolated from LCM-retrieved endothelial tissue and processed for RNA-seq.

2025-08-04 | GSE296511 | GEO

An optimized workflow for transcriptomic analysis from archival paraformaldehyde-fixed retinal tissues collected by laser capture microdissection

Project description:RNA sequencing (RNA-seq) coupled with laser capture microdissection (LCM) is a powerful tool for transcriptomic analysis in unfixed fresh-frozen tissues. Fixation of ocular tissues for immunohistochemistry commonly involves the use of paraformaldehyde (PFA) followed by embedding in Optimal Cutting Temperature (OCT) medium for long-term cryopreservation. However, the quality of RNA derived from such archival PFA-fixed/OCT-embedded samples is often compromised, limiting its suitability for transcriptomic studies. In this study, we aimed to develop a methodology to extract high-quality RNA from PFA-fixed canine eyes by utilizing LCM to isolate retinal tissue. We demonstrate the efficacy of an optimized LCM and RNA purification protocol for transcriptomic profiling of PFA-fixed retinal specimens. We compared four pairs of canine retinal tissues, where one eye was subjected to PFA-fixation prior to OCT embedding, while the contralateral eye was embedded fresh frozen (FF) in OCT without fixation. Since the mRNA obtained from PFA-fixed retinas were contaminated with genomic DNA, we employed two rounds of DNase I treatment to obtain RNA suitable for RNA-seq. Notably, the quality of sequencing reads and gene sets identified from both PFA-fixed and FF tissues were nearly identical. In summary, our study introduces an optimized workflow for transcriptomic profiling from PFA-fixed archival retina. This refined methodology paves the way for improved transcriptomic analysis of preserved ocular tissue, bridging the gap between optimal sample preservation and high-quality RNA data acquisition.

2024-05-15 | GSE262625 | GEO

An optimized workflow for transcriptomic analysis from archival paraformaldehyde-fixed retinal tissues collected by laser capture microdissection

Project description:An optimized workflow for transcriptomic analysis from archival paraformaldehyde-fixed retinal tissues collected by laser capture microdissection

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Laser Capture Microdissection (LCM) analysis of mycorrhizal rice roots

Project description:Laser Capture Microdissection (LCM) analysis of mycorrhizal rice roots

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Laser Capture Microdissection and RNA Sequencing Human Adamantinomatous Craniopharyngioma

Project description:Laser Capture Microdissection and RNA Sequencing Human Adamantinomatous Craniopharyngioma

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Comparison between conventionally-isolated and laser capture microdissected glomeruli

Project description:We compared the gene expression analysis of 2 different glomerular isolation techniques (laser capture microdissection with 2 rounds of RNA amplification and unamplified glomerular RNA after iron perfusion with glomerular sieving) and obtained different results depending on the glomerular isolation technique that was used Keywords: time course

2007-09-11 | GSE8998 | GEO

Laser capture microdissection of endothelial and neuronal cells from human dorsolateral prefrontal cortex

Project description:We used laser capture microdissection to isolate both microvascular endothelial cells and neurons from post mortem brain tissue from patients with schizophrenia and bipolar disorder and healthy controls. RNA was isolated from these cell populations, amplified, and analysed using Affymetrix HG133plus2.0 GeneChips. In the first instance, we used the dataset to compare the neuronal and endothelial data, in order to demonstrate that the predicted differences between cell types could be detected using this methodology. Keywords: cell type comparison, laser capture microdissection

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Identification of Inner Mucus-Associated Bacteria by Laser Capture Microdissection

Project description:Identification of Inner Mucus-Associated Bacteria by Laser Capture Microdissection

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Tissue-specific laser microdissection of the Brassica napus funiculus improves gene identification and spatial control of biological processes

Project description:Tissue-specific RNA profiling of the Brassica napus funiculus and funicular subcompartments using laser microdissection and RNA-sequencing

2015-08-20 | GSE71859 | GEO

Laser Capture Microdissection and RNA Sequencing Human Adamantinomatous Craniopharyngioma

Project description:Laser capture microdissection of cluster cells, palisading epithelium and glial tissue from 2 cases of human adamantinomatous craniopharyngioma. 1 case performed in duplicate.

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