Laser Capture Microdissection for Protein and NanoString RNA Analysis

Golubeva, Yelena; Salcedo, Rosalba; Mueller, Claudius; Liotta, Lance A.; Espina, Virginia

doi:10.1007/978-1-62703-056-4_12

Cited by 49 publications

(29 citation statements)

References 35 publications

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“…Furthermore, we analyzed IL-15 and IL-15Rα mRNA levels in the islets from T1D patients, autoantibody positive, and normal donors. The islets were microdissected based on insulin positivity using laser capture microscopy (LCM) (29). Consistent with the immunohistochemistry results, we found higher IL-15 ( Fig.…”

Section: Inhibiting Il-15 Signaling At the Prediabetic Stage Delayed supporting

confidence: 38%

Insulin-dependent diabetes induced by pancreatic beta cell expression of IL-15 and IL-15Rα

Chen

Feigenbaum

Awasthi

et al. 2013

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

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Increased serum levels of IL-15 are reported in type 1 diabetes (T1D). Here we report elevated serum soluble IL-15Rα levels in human T1D. To investigate the role of IL-15/IL-15Rα in the pathogenesis of T1D, we generated double transgenic mice with pancreatic β-cell expression of IL-15 and IL-15Rα. The mice developed hyperglycemia, marked mononuclear cell infiltration, β-cell destruction, and anti-insulin autoantibodies that mimic early human T1D. The diabetes in this model was reversed by inhibiting IL-15 signaling with anti-IL2/IL15Rβ (anti-CD122), which blocks IL-15 transpresentation. Furthermore, the diabetes could be reversed by administration of the Janus kinase 2/3 inhibitor tofacitinib, which blocks IL-15 signaling. In an alternative diabetes model, nonobese diabetic mice, IL15/IL-15Rα expression was increased in islet cells in the prediabetic stage, and inhibition of IL-15 signaling with anti-CD122 at the prediabetic stage delayed diabetes development. In support of the view that these observations reflect the conditions in humans, we demonstrated pancreatic islet expression of both IL-15 and IL-15Rα in human T1D. Taken together our data suggest that disordered IL-15 and IL-15Rα may be involved in T1D pathogenesis and the IL-15/IL15Rα system and its signaling pathway may be rational therapeutic targets for early T1D.

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Section: Inhibiting Il-15 Signaling At the Prediabetic Stage Delayed supporting

confidence: 38%

Insulin-dependent diabetes induced by pancreatic beta cell expression of IL-15 and IL-15Rα

Chen

Feigenbaum

Awasthi

et al. 2013

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

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“…The procedures were carried out according to the manufacturer's instructions. The whole process was completed within 30 min [45].…”

Section: Quick Stainingmentioning

confidence: 99%

Transcriptome profiling of laser-captured germ cells and functional characterization of zbtb40 during 17alpha-methyltestosterone-induced spermatogenesis in orange-spotted grouper (Epinephelus coioides)

Yang

Zhong

et al. 2020

BMC Genomics

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Background: Spermatogenesis is an intricate process regulated by a finely organized network. The orange-spotted grouper (Epinephelus coioides) is a protogynous hermaphroditic fish, but the regulatory mechanism of its spermatogenesis is not well-understood. In the present study, transcriptome sequencing of the male germ cells isolated from orange-spotted grouper was performed to explore the molecular mechanism underlying spermatogenesis. Results: In this study, the orange-spotted grouper was induced to change sex from female to male by 17alphamethyltestosterone (MT) implantation. During the spermatogenesis, male germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa) were isolated by laser capture microdissection. Transcriptomic analysis for the isolated cells was performed. A total of 244,984,338 clean reads were generated from four cDNA libraries. Real-time PCR results of 13 genes related to sex differentiation and hormone metabolism indicated that transcriptome data are reliable. RNA-seq data showed that the female-related genes and genes involved in hormone metabolism were highly expressed in spermatogonia and spermatozoa, suggesting that these genes participate in the spermatogenesis. Interestingly, the expression of zbtb family genes showed significantly changes in the RNA-seq data, and their expression patterns were further examined during spermatogenesis. The analysis of cellular localization of Eczbtb40 and the co-localization of Eczbtb40 and Eccyp17a1 in different gonadal stages suggested that Eczbtb40 might interact with Eccyp17a1 during spermatogenesis. Conclusions:Our study, for the first time, investigated the transcriptome of the male germ cells from orangespotted grouper, and identified functional genes, GO terms, and KEGG pathways involved in spermatogenesis. Furthermore, Eczbtb40 was first characterized and its role during spermatogenesis was predicted. These data will contribute to future studies on the molecular mechanism of spermatogenesis in teleosts.

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“…The ArcturusXT system used for the LCM in the present study is the most advanced microdissection equipment currently available and combines the technologies of LCM and ultraviolet laser dissection . This system is equipped with ultraviolet and near‐infrared lasers.…”

Section: Discussionmentioning

confidence: 99%

Laser capture microdissection for detecting the expression of epithelial–mesenchymal transition‐related genes in epithelial and spindle cells of paraffin‐embedded formalin‐fixed biphasic synovial sarcoma

Wang

Liu

Zou

et al. 2018

Clin Exp Pharma Physio

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Synovial sarcoma (SS) is a mesenchymal malignant neoplasm showing characteristics of epithelial-mesenchymal biphasic differentiation. SS is of uncertain cellular origin; however, studies have suggested that SS originates from a somatic stem cell population. In this study, we aim to determine whether differential morphological features of the epithelial-mesenchymal transition (EMT) contributed to the tumourigenesis of SS invasion and metastasis. Twelve paraffin-embedded formalin-fixed tissue (FFPE) SS tissue specimens were obtained, and laser capture microdissection (LCM) with the ArcturusXT system and small chip method (SCM) were used to isolate and purify spindle and epithelial cells from SS specimens. The TRIzol method was used to extract RNA, and the mRNA levels of EMT-related genes in epithelial and spindle cells of SS specimens were measured using real-time fluorescent quantitative reverse transcription polymerase chain reaction (qRT-PCR). The results show that collection of about 2 × 10 cells from FFPE samples using LCM was sufficient for qRT-PCR, with an efficiency of 75%. Compared with LCM, 72.2% (13 of 18) RNA samples were successfully extracted using SCM to isolate cells from FFPE SS tissues. In the 16 samples (11 spindle cell samples and 5 epithelial cell samples), Snail mRNA was significantly upregulated in spindle cell areas compared with that in epithelial cell areas (P = .001). Expression levels of the epithelial marker E-cadherin and the mesenchymal marker N-cadherin were not significantly different between epithelial and spindle cell areas. In spindle cells of recurrent SS samples, the mRNA levels of E-cadherin, N-cadherin, Snail, and Slug were higher in primary SS samples than in recurrent samples. Taken together, our results indicated that in SS samples, Snail mRNA was upregulated in spindle cell areas compared with that in epithelial cell areas and that the expression of EMT-related genes was increased in primary SS. LCM could be used to isolate and purify RNA from FFPE samples.

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Laser Capture Microdissection for Protein and NanoString RNA Analysis

Cited by 49 publications

References 35 publications

Insulin-dependent diabetes induced by pancreatic beta cell expression of IL-15 and IL-15Rα

Insulin-dependent diabetes induced by pancreatic beta cell expression of IL-15 and IL-15Rα

Transcriptome profiling of laser-captured germ cells and functional characterization of zbtb40 during 17alpha-methyltestosterone-induced spermatogenesis in orange-spotted grouper (Epinephelus coioides)

Laser capture microdissection for detecting the expression of epithelial–mesenchymal transition‐related genes in epithelial and spindle cells of paraffin‐embedded formalin‐fixed biphasic synovial sarcoma

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