Gene expression patterns in rat dentate granule cells: comparison between fresh and fixed tissue

Qin, Yongjun; Heine, Vivi M.; Karst, H.; Lucassen, Paul J.; Joëls, Marian

doi:10.1016/j.jneumeth.2003.08.010

Cited by 10 publications

(6 citation statements)

References 30 publications

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“…First, the method of tissue storage was assessed for the preservation of high-quality RNA in cervical biopsy specimens. In agreement with previous studies [2,3,32], our data strongly suggested that FR cervical tissue was optimal for obtaining a high quality and quantity of RNA (Fig. 1A), whereas FFPE tissue processing caused considerable degradation and low yield, possibly as a result of nucleic acid crosslinking with proteins, covalent modifications, and strand breaks [33].…”

Section: Discussionsupporting

confidence: 92%

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

DeCarlo

Escott

Werner

et al. 2008

Analytical Biochemistry

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Optimal sample handling techniques for tissue preparation and storage, RNA extraction and quantification, and target gene detection are crucial for reliable gene expression analysis. Methods for measuring low-expressing genes, such as interferons, in human cervical samples are not described in the scientific literature. To detect interferon mRNA in human cervical samples we obtained normal and dysplastic frozen and formalin-fixed cervical biopsies from colposcopy. Histopathological diagnosis was performed by one pathologist. Cervical keratinocytes were isolated using laser capture microdissection. Immortalized keratinocytes transduced with or devoid of an HPV oncogene were used for initial method development. RNA from samples was extracted and integrity tested to compare tissue storage and extraction methods. The expression of five housekeeping genes was analyzed in cell lines and patient tissue to permit normalization between samples using quantitative real-time polymerase chain reaction. The usefulness of cDNA amplification was assessed for the detection of low-expressing interferon κ in cervical tissue. Here we report optimal tissue storage conditions, RNA extraction, sample normalization, and transcript amplification, as well as the sensitivity of quantitative real-time polymerase chain reaction and laser capture microdissection, for interferon κ detection in cervical tissue. Without these optimized techniques, interferon κ detection would be unattainable in cervical samples. KeywordsCervical keratinocytes; Frozen and formalin-fixed cervical tissue; Housekeeping genes; Interferons; Interferon κ; Laser capture microdissection; Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) 1 for the molecular analysis of disease is a powerful and widely used tool. Extraction of high-quality RNA for use in gene expression analysis techniques such as reverse transcription (RT), qRT-PCR, and cDNA microarrays is of great importance. Although obtaining high-quality RNA from cell lines is relatively straightforward, the complexity and heterogeneity of human tissue pose considerable challenges for linking gene expression patterns with disease state. Nonetheless,

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Section: Discussionsupporting

confidence: 92%

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

DeCarlo

Escott

Werner

et al. 2008

Analytical Biochemistry

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“…However, RNA degradation is a major drawback in most common fixation protocols recommended for tissue processing prior to LCM [30]. Therefore, specific fixation and RNA isolation protocols have been developed for LCM [17], [31].…”

Section: Methodsmentioning

confidence: 99%

A New and Reliable Guide for Studies of Neuronal Loss Based on Focal Lesions and Combinations of In Vivo and In Vitro Approaches

et al. 2013

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In this study, we describe a simple and reliable method to study neuroprotective effects in living and organized neural tissue. This method, which was based on retinal explants for in vivo focal lesions, was conceived as a collection of modular procedures, which can be customized for particular demands. With this model, it is possible to combine immunohistochemistry with image data analysis to track the two- or three-dimensional redistribution of proteins as a time/space function of primary cell loss. At the same time, it is possible to finely control the exposure of the tissue to specific drugs and molecules. In order to illustrate the use of the proposed method, we tested the effects of two different nanotube compounds on retinal explant viability. Transcriptome analyses can be separately performed in the lesion focus and penumbra with laser capture microdissection followed by polymerase chain reaction analyses. In addition, other common experimental drawbacks, such as high individual variance, are eliminated. With intraocular injections, treatments can be verified in vivo, with one eye serving as the experimental tissue and the other serving as the control tissue. In summary, we describe a flexible and easy method, which can be useful in combination with a broad variety of recently developed neuroprotective strategies, to study neurodegeneration.

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“…Similarly, for brain samples, fixation by ethanol is superior to formalin for preserving RNA integrity suitable for expression profiling of brain tissues by LCM [ 27 ]. Qin et al [ 28 ] have previously demonstrated that a fresh frozen and ethanol fixed hippocampal DG granule cell sample yielded a transcript expression profile comparable to that of a non-fixed cell sample, and was more reliable compared to the RNA retrieved from paraformaldehyde-fixed, paraffin embedded tissue sample. Therefore, ethanol is considered to be a suitable fixation solution for fresh frozen brain tissues although staining effects on brain samples have not been previously reported.…”

Section: Discussionmentioning

confidence: 99%

Application of NeuroTrace staining in the fresh frozen brain samples to laser microdissection combined with quantitative RT-PCR analysis

et al. 2015

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BackgroundThe heterogeneity of the brain requires appropriate molecular biological approaches to account for its morphological complexity. Laser-assisted microdissection followed by transcript profiling by quantitative determination has been reported to be an optimal methodology. Nevertheless, not all brain regions can be identified easily without staining, restricting the accuracy and efficiency in sampling. The aim of the present study was to validate whether fixation and staining treatments are suitable for quantitative transcript expression analysis in laser microdissection (LMD) samples. Quantitative RT-PCR was used to determine the absolute transcript expression levels and profiles of samples obtained from the hippocampal dentate gyrus from fresh frozen mice brain sections that had been fixed with ethanol and stained with NeuroTrace. The results were compared with those obtained from unfixed and unstained samples.ResultsWe found that the quantitative relationship of transcript expression levels between various housekeeping genes and immediate early genes was preserved, although the preparation compromised the yield of the transcripts. In addition, histological and molecular integrities of the fixed and stained specimens were preserved for at least a week at room temperature. Based on the lobe specific profiles of transcripts in the anterior and posterior lobes of the pituitary, we confirmed that no cross-contamination on transcription expressions occurred as a result of the fixation and staining.ConclusionsWe have provided detailed information of the procedures on ethanol fixation followed by NeuroTrace staining on the absolute quantitative RT-PCR analysis using microdissected fresh frozen mouse brain tissues. The present study demonstrated that quantitative transcript expression analysis can be conducted reliably on stained tissues. This method is suitable for applications in basic and clinical studies on particular transcript expressions in various regions of the brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1222-9) contains supplementary material, which is available to authorized users.

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Gene expression patterns in rat dentate granule cells: comparison between fresh and fixed tissue

Cited by 10 publications

References 30 publications

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

A New and Reliable Guide for Studies of Neuronal Loss Based on Focal Lesions and Combinations of In Vivo and In Vitro Approaches

Application of NeuroTrace staining in the fresh frozen brain samples to laser microdissection combined with quantitative RT-PCR analysis

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