Combining laser capture microdissection with quantitative real-time PCR: Effects of tissue manipulation on RNA quality and gene expression

Kerman, Ilan A.; Buck, Bradley J.; Evans, Simon J.; Akil, Huda; Watson, Stanley J.

doi:10.1016/j.jneumeth.2005.10.010

Cited by 72 publications

(62 citation statements)

References 41 publications

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“…To date, few studies using LCM have examined the quality of the isolated RNA; however, a recent study reported that the RNA isolated from LCM of stained cerebellum had an RNA integrity number of ϳ5 (49). Although we had some RNA degradation, this was probably due to staining, which has been shown to decrease RNA quality (49). Since staining is essential for cell localization, some RNA degradation is inevitable.…”

Section: Resultsmentioning

confidence: 94%

“…Consequently, the gene profiles that we generated (see below) were reflective of cells in their natural state. We used the RNA integrity number to assess the RNA quality since this has been shown to be the method of choice when evaluating RNA integrity from LCM samples (49). Using the RNA integrity number scale, which ranges from 10 (ideal intact RNA) to 1 (totally degraded RNA (38)), we routinely obtained values ϳ7.5 and ϳ6.6 for limbal and corneal RNAs, respectively.…”

Section: Resultsmentioning

confidence: 99%

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Transcriptional Profiling of Enriched Populations of Stem Cells Versus Transient Amplifying Cells

Zhou¹,

Lavker³

2006

Journal of Biological Chemistry

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The basal layer of limbal and central corneal epithelium is enriched in stem cells and transient amplifying cells, respectively. This physical separation of stem and transient amplifying cells makes the limbal/corneal epithelium an exceptionally suitable system for isolating basal cells enriched in these two proliferative populations. Prior attempts to isolate epithelial stem cells used methods such as proteolytic tissue dissociation and cell sorting that could potentially alter their gene expression profile. Using laser capture microdissection, we were able to isolate resting limbal and corneal basal cells from frozen sections with minimal tissue processing, thereby improving the yield and quality of RNA. Analyses of RNA isolated from 300 limbal and corneal basal cells from eight mice revealed a set of ϳ100 genes that are differentially expressed in limbal cells versus corneal epithelial basal cells. Semiquantitative reverse transcription-PCR confirmed the up-regulation of three limbal and three corneal genes. LacZ identification of epiregulin from epiregulin-null mice and immunohistochemical staining of wild type mice confirmed that epiregulin, one of the limbal epithelium-enriched genes, was associated with the limbal epithelial basal cells. Within the limbal and corneal basal cells, we detected previously unknown genes that were differentially expressed in these two regions that contribute further to our understanding of the unique heterogeneity of these two closely related basal cell populations. Our findings indicate that we can obtain accurate gene expression profiles of the stem cell-enriched limbal basal cell population in their "natural" quiescent state.The ability to identify, purify, and characterize epithelial stem cells is a critically important issue in epithelial stem cell biology as it will further our understanding on how stem cells are regulated. Of the various epithelial tissues studied, the limbal/corneal epithelium has yielded a great deal of information about stem cells (1). Based on a combination of (i) keratin expression data (2); (ii) in vivo and in vitro cell kinetic data (3-7); (iii) centripetal migration studies (8 -13); and (iv) corneal regeneration and reconstitution studies (14 -20), it is well accepted that the corneal epithelial stem cells are preferentially located in the limbal epithelial basal layer (for reviews, see Refs. 1 and 21-27). Furthermore, since the limbal stem cells can be millimeters away from their central corneal progeny transient amplifying (TA) 2 cells (1,2,4,7,24,28), the limbal/corneal system is ideally suited for isolating relatively pure populations of epithelial stem and TA cells for subsequent analyses.A lack of stem cell-specific markers has been an impediment for the isolation and subsequent biological and biochemical characterization of epithelial stem cells. Recently, transcriptional profiling of enriched populations of putative epithelial stem cells has been used to search for epithelial stem cellspecific markers. Methods used to isolate the putativ...

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Transcriptional Profiling of Enriched Populations of Stem Cells Versus Transient Amplifying Cells

Zhou¹,

Lavker³

2006

Journal of Biological Chemistry

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“…Sections from these series were also used for mRNA in situ hybridization. The cresyl violet step was omitted to preserve RNA quality (33). Care was taken to ensure that the same level of the dentate gyrus (Bregma = −3.30) was taken from each rat (34).…”

Section: Methodsmentioning

confidence: 99%

Fibroblast growth factor-2 (FGF2) augmentation early in life alters hippocampal development and rescues the anxiety phenotype in vulnerable animals

Turner

Clinton

Thompson

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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112

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Individuals with mood disorders exhibit alterations in the fibroblast growth factor system, including reduced hippocampal fibroblast growth factor-2 (FGF2). It is difficult, however, to pinpoint whether these alterations are a cause or consequence of the disorder. The present study asks whether FGF2 administered the day after birth has long-lasting effects on hippocampal development and emotionality. We show that early-life FGF2 shifts the pace of neurogenesis, with an early acceleration around weaning followed by a deceleration in adulthood. This, in turn, results in a denser dentate gyrus with more neurons. To assess the impact of early-life FGF2 on emotionality, we use rats selectively bred for differences in locomotor response to novelty. Selectively bred low-responder (bLR) rats show low levels of novelty-induced locomotion and exhibit high levels of anxiety-and depression-like behavior compared with their selectively bred high-responder counterparts. Early-life FGF2 decreased anxiety-like behavior in highly anxious bLRs without altering other behaviors and without affecting high-responder rats. Laser capture microscopy of the dentate gyrus followed by microarray analysis revealed genes that were differentially expressed in bLRs exposed to early-life FGF2 vs. vehicle-treated bLRs. Some of the differentially expressed genes that have been positively associated with anxiety were down-regulated, whereas genes that promote cell survival were up-regulated. Overall, these results show a key role for FGF2 in the developmental trajectory of the hippocampus as well as the modulation of anxiety-like behavior in adulthood, and they point to potential downstream targets for the treatment of anxiety disorders. elevated plus maze | gene expression microarray H uman postmortem studies suggest a role for the fibroblast growth factor (FGF) system in the pathophysiology of mood disorders. The expression of several members of the FGF family was altered in the forebrain of patients with major depressive disorder (MDD). Specifically, FGF2 mRNA expression was down-regulated in cortical areas and the hippocampus in MDD compared with controls (1, 2). The hippocampus seems to be particularly affected in MDD (3). A potential role for FGF2 in depression and anxiety disorders was first suggested by pharmacological studies in rodents, which showed that administration of antidepressant and anxiolytic drugs can increase FGF2 expression in the hippocampus (3-6). We then asked whether FGF2 is a modulator of emotionality in rodents and discovered that repeated administration of FGF2 was both antidepressant and anxiolytic (7,8).Using rats selectively bred for differences in novelty-seeking, we found that hippocampal expression of FGF2 mRNA was higher in high responders to novelty (bHRs) than in the low responders to novelty (bLRs). The bHRs have higher FGF2 levels and exhibit less anxiety-like behavior, whereas the bLRs exhibit more anxiety-like behavior. FGF2 seems to be a protective factor against anxiety-like behavior in the adult (7). Th...

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“…The 28S/18S rRNA ratio was used to assess RNA quality, using the respective areas under the 28S and the 18S peaks (Kerman et al, 2006).…”

Section: Obtaining Cdnamentioning

confidence: 99%

Functional autoradiography and gene expression analysis applied to the characterization of the α2-adrenergic system in the chicken brain

Dı́ez-Alarcia

Mostany

Dos-Anjos

et al. 2009

Journal of Chemical Neuroanatomy

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Combining laser capture microdissection with quantitative real-time PCR: Effects of tissue manipulation on RNA quality and gene expression

Cited by 72 publications

References 41 publications

Transcriptional Profiling of Enriched Populations of Stem Cells Versus Transient Amplifying Cells

Transcriptional Profiling of Enriched Populations of Stem Cells Versus Transient Amplifying Cells

Fibroblast growth factor-2 (FGF2) augmentation early in life alters hippocampal development and rescues the anxiety phenotype in vulnerable animals

Functional autoradiography and gene expression analysis applied to the characterization of the α2-adrenergic system in the chicken brain

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