Application of laser-capture microdissection to analysis of gene expression in the testis

The isolation and molecular analysis of highly purified cell populations from complex, heterogeneous tissues has been a challenge for many years. Spermatogenesis in the testis is a particularly difficult process to study given the unique multiple cellular associations within the seminiferous epithelium, making the isolation of specific cell types difficult. Laser-capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. This technology has enhanced our ability to directly examine gene expression in enriched testicular cell populations by routine methods of gene expression analysis, such as real-time RT-PCR, differential display, and gene microarrays. The application of LCM has however introduced methodological hurdles that have not been encountered with more conventional molecular analyses of whole tissue. In particular, tissue handling (i.e. fixation, storage, and staining), consumables (e.g. slide choice), staining reagents (conventional H&E vs. fluorescence), extraction methods, and downstream applications have all required re-optimisation to facilitate differential gene expression analysis using the small amounts of material obtained using LCM. This review will discuss three critical issues that are essential for successful procurement of cells from testicular tissue sections; tissue morphology, capture success, and maintenance of molecular integrity. The importance of these issues will be discussed with specific reference to the two most commonly used LCM systems; the Arcturus PixCell IIe and PALM systems. The rat testis will be used as a model, and emphasis will be placed on issues of tissue handling, processing, and staining methods, including the application of fluorescence techniques to assist in the identification of cells of interest for the purposes of mRNA expression analysis.     

TRAP-rc,Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genome-wide studies.     

A manual method for the purification of fluorescently labeled neurons from the mammalian brain

Sorting of fluorescent cells is a powerful technique for revealing the cellular processes that differ among the various cell types found in complex tissues. With the recent availability of transgenic mouse strains in which specific subpopulations of neurons are labeled, it has become desirable to purify these fluorescent neurons from their surrounding hetereogeneous brain tissue for electrophysiological, biochemical and molecular analyses. This has been accomplished using automated fluorescence-activated cell sorting (FACS) and laser capture microdissection (LCM). Although these procedures can be effective, they have some serious disadvantages, including high equipment costs and difficulty in obtaining samples completely free of contaminating tissue. Here we offer an alternative protocol for purifying fluorescent neurons, which relies on less-expensive equipment, readily produces perfectly pure samples and can be applied to neurons that are only dimly labeled and present in low numbers. The entire protocol can be completed in 3-5 h.     

Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription

The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression.     

Long-chain metabolites of vitamin E: Interference with lipotoxicity via lipid droplet associated protein PLIN2

The long-chain metabolites of vitamin E (LCM) emerge as a new class of regulatory metabolites and have been considered as the active compounds formed during vitamin E metabolism. The bioactivity of the LCM is comparable to the already established role of other fat-soluble vitamins. The biological modes of action of the LCM are far from being unraveled, but first insights pointed to distinct effects and suggested a specific receptor, which in turn lead to the aforementioned hypothesis. Here, a new facet on the interaction of LCM with foam cell formation of THP-1 macrophages is presented. We found reduced levels of mRNA and protein expression of lipid droplet associated protein PLIN2 by α-tocopherol (α-TOH), whereas the LCM and the saturated fatty acid, stearic acid, increased expression levels of PLIN2. In a lipotoxic setup (0–800?μM stearic acid and 0–100?μM α-TOH or 0–5?μM α-13′-COOH) differences in cellular viability were found. A reduced viability was observed for cells under co-treatment of α-TOH and stearic acid, whereas an increased viability for stearic acid incubation in combination with α-13′-COOH was observed. The striking similarity of PLIN2 expression levels and worsened or mitigated lipotoxicity, respectively, revealed a protective effect of PLIN2 on basal stearic acid-induced lipotoxic conditions in PLIN2 knockdown experiments. Based on our results, we conclude that α-13′-COOH protects cells from lipotoxicity, at least partially via PLIN2 regulation.Herewith another facet of LCM functionality was presented and their reputation as regulatory metabolites was further established.     

TRAP1 Shows Clinical Significance and Promotes Cellular Migration and Invasion through STAT3/MMP2 Pathway in Human Esophageal Squamous Cell Cancer

Tumor necrosis factor receptor-associated protein 1 (TRAP1), an important member of mitochondrial heat shock protein 90 family, is involved in multiple biological processes in several types of tumors. However, its pathological role in esophageal squamous cell cancer (ESCC) remains unknown. Herein, we demonstrated the clinical value of TRAP1, and its role in apoptosis and motility in ESCC. The clinical potential of TRAP1 was investigated through immunohistochemical analysis in 328 ESCC samples, which revealed that strong TRAP1 expression was associated with increased risk of lymph node metastasis, while high TRAP1 expression correlated with poor prognosis. Expression of TRAP1 was found to be an independent prognostic factor for patients with ESCC. Additionally, the upregulation of TRAP1 antagonized cisplatin-induced apoptosis while its downregulation sensitized cells to cisplatin-induced apoptosis. As revealed by the transwell assay, TRAP1 overexpression promoted cellular migration and invasion as compared to the control groups. In contrast, silencing of endogenous TRAP1 expression attenuated the ability of migration and invasion. Finally, the molecular mechanism investigated in the present study demonstrated that TRAP1-mediated migration and invasion occurred through STAT3/MMP2 signaling pathway. In conclusion, TRAP1 may be considered as a molecular predictive marker for prognosis and a novel molecular candidate for therapeutic target in ESCC.     

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.     

Evidence for the complexity of microRNA-mediated regulation in ovarian cancer: a systems approach

MicroRNAs (miRNAs) are short (～22 nucleotides) regulatory RNAs that can modulate gene expression and are aberrantly expressed in many diseases including cancer. Previous studies have shown that miRNAs inhibit the translation and facilitate the degradation of their targeted messenger RNAs (mRNAs) making them attractive candidates for use in cancer therapy. However, the potential clinical utility of miRNAs in cancer therapy rests heavily upon our ability to understand and accurately predict the consequences of fluctuations in levels of miRNAs within the context of complex tumor cells. To evaluate the predictive power of current models, levels of miRNAs and their targeted mRNAs were measured in laser captured micro-dissected (LCM) ovarian cancer epithelial cells (CEPI) and compared with levels present in ovarian surface epithelial cells (OSE). We found that the predicted inverse correlation between changes in levels of miRNAs and levels of their mRNA targets held for only ～11% of predicted target mRNAs. We demonstrate that this low inverse correlation between changes in levels of miRNAs and their target mRNAs in vivo is not merely an artifact of inaccurate miRNA target predictions but the likely consequence of indirect cellular processes that modulate the regulatory effects of miRNAs in vivo. Our findings underscore the complexities of miRNA-mediated regulation in vivo and the necessity of understanding the basis of these complexities in cancer cells before the therapeutic potential of miRNAs can be fully realized.     

MicroRNA-125a is over-expressed in insulin target tissues in a spontaneous rat model of Type 2 Diabetes

Background

The methods used for sample selection and processing can have a strong influence on the expression values obtained through microarray profiling. Laser capture microdissection (LCM) provides higher specificity in the selection of target cells compared to traditional bulk tissue selection methods, but at an increased processing cost. The benefit gained from the higher tissue specificity realized through LCM sampling is evaluated in this study through a comparison of microarray expression profiles obtained from same-samples using bulk and LCM processing.

Methods

Expression data from ten lung adenocarcinoma samples and six adjacent normal samples were acquired using LCM and bulk sampling methods. Expression values were evaluated for correlation between sample processing methods, as well as for bias introduced by the additional linear amplification required for LCM sample profiling.

Results

The direct comparison of expression values obtained from the bulk and LCM sampled datasets reveals a large number of probesets with significantly varied expression. Many of these variations were shown to be related to bias arising from the process of linear amplification, which is required for LCM sample preparation. A comparison of differentially expressed genes (cancer vs. normal) selected in the bulk and LCM datasets also showed substantial differences. There were more than twice as many down-regulated probesets identified in the LCM data than identified in the bulk data. Controlling for the previously identified amplification bias did not have a substantial impact on the differences identified in the differentially expressed probesets found in the bulk and LCM samples.

Conclusion

LCM-coupled microarray expression profiling was shown to uniquely identify a large number of differentially expressed probesets not otherwise found using bulk tissue sampling. The information gain realized from the LCM sampling was limited to differential analysis, as the absolute expression values obtained for some probesets using this study's protocol were biased during the second round of amplification. Consequently, LCM may enable investigators to obtain additional information in microarray studies not easily found using bulk tissue samples, but it is of critical importance that potential amplification biases are controlled for.     

A Tie2‐driven BAC‐TRAP transgenic line for in vivo endothelial gene profiling

Recent technological innovations including bacterial artificial chromosome‐based translating ribosome affinity purification (BAC‐TRAP) have greatly facilitated analysis of cell type‐specific gene expression in vivo, especially in the nervous system. To better study endothelial gene expression in vivo, we have generated a BAC‐TRAP transgenic mouse line where the L10a ribosomal subunit is tagged with EGFP and placed under the control of the endothelium‐specific Tie2 (Tek) promoter. We show that transgene expression in this line is widely, but specifically, detected in endothelial cells in several brain regions throughout pre‐ and postnatal development, as well as in other organs. We also show that this line results in highly significant enrichment of endothelium‐specific mRNAs from brain tissues at different stages. This BAC‐TRAP line therefore provides a useful genetic tool for in vivo endothelial gene profiling under various developmental, physiological, and pathological conditions. genesis 54:136–145, 2016. © 2016 Wiley Periodicals, Inc.