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1 Second Department of Pathology, Wakayama Medical University, Kimiidera 811-1, Wakayama City, Wakayama, 641-0012, Japan
2 Department of Geriatric Medicine, Kanazawa Medical University, Uchinada 1-1, Ishikawa, Ishikawa, 920-0290, Japan
3 Department of Pediatrics, Wakayama Medical University, Kimiidera 811-1, Wakayama City, Wakayama, 641-0012, Japan
4 The Cancer Institute of New Jersey, UMDNJ-Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, New Jersey 08903, USA
(Requests for offprints should be addressed to M Nakamura; Email: marumisa{at}wakayama-med.ac.jp)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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There is significant interest in analyzing gene expression of distinct cell populations. Heterogeneous populations of cells within tissues of various types possess correspondingly different patterns of gene expression, and these cell types must be separated from one another for accurate assessment of gene expression. Tong et al.(1994) has reported that a microisolation system using a micromanupilator tool was applied for mRNA phenotyping of a blood cell lineage. Laser capture microdissection (LCM) is a particularly useful tool for recovering small cell samples and even enables the collection of individual cells from tissue sections (Emmert-Buck et al. 1996). This method facilitates the separation of histologically distinct cells so that proteins, DNA or RNA from these cells can be analyzed in isolation from the surrounding cells (Bonner et al. 1997). Osteoclasts act centrally in the remodeling of bone in normal and diseased states. Nonetheless, because of their low numbers within bone, cell culture model systems have been increasingly used to investigate the biochemical functions of osteoclasts (Udagawa et al.1989, Nakamura et al. 1998). However, because of their heterogeneity and adherence to the plate in such systems, there has been difficulty and controversy in analyzing these cell types. Thus, a more sensitive isolation method for osteoclasts is needed.
To address this problem, we used LCM techniques to isolate a pure population of osteoclast-like cells. We then analyzed RAMP gene expression in microdissected osteoclast-like cells using RT-PCR.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Osteoclast differentiation in vitro was induced using the technique described by Udagawa et al.(1989). Both bone marrow cells and spleen cells were obtained from 10-to 14-week-old male C57BL/6 mice (Charles River, Sagamihara, Japan). The bone marrow cells were collected from tibiae and femora. Splenic tissue was cut with scissors and dispersed by pipetting, then the spleen cells were collected by centrifugation at 1000 r.p.m. for 5 min at 4 °C. Bone marrow cells were co-cultured with spleen cells (2 x 106 cells/ml for each cell type) on a film produced for use in LCM (Matsunami Glass Co., Osaka, Japan) for 10 days at 37 °C in a humidified atmosphere of 5% CO2. Cultures were fed with 
-modified Eagle’s medium supplemented with penicillin and streptomycin, 10% fetal calf serum (Hyclone, Logan, UT, USA) and 10–8 M 1,25(OH)2 D3 (Calbiochem-Novabiochem Co., San Diego, CA, USA). Multinucleated osteoclast-like cells were then isolated using LCM. All the animal experimental procedures were approved by the Animal Care and Use Committee of Wakayama Medical University (Wakayama, Japan).
LCM of samples
Before LCM, cells were fixed in ethanol for 1 min and stained for 3 min with filtered hematoxylin. They were then washed with sterilized water and air-dried for 10 min. LCM of cultured osteoclast-like cells was performed using the Application Solutions Laser Micro-dissection System (Leica Microsystems Co., Tokyo, Japan) according to the manufacturer’s instructions.
RNA isolation
Total RNA was extracted from 250 LCM-captured cells and 250 spleen cells. The spleen cells used for RNA extraction were from an aliquot of those prepared for the co-culture system. Total RNA extraction was performed using TRIzol LS Reagent (Invitrogen Life Technologies Co., Carlsbad, CA, USA) as described by the manufacturer. Briefly, 170 µl TRIzol reagent was added to a tube containing LCM cells and this was incubated for 5 min at room temperature. Forty microliters of chloroform were then added and the tube was incubated at room temperature for a further 15 min. The samples were then centrifuged at 12 000 g for 15 min. The aqueous phase was transferred to a new tube and isopropyl alcohol was added followed by centrifugation at 12 000 g for 10 min. The RNA precipitate was washed with 70% ethanol and dissolved in 20 µl sterilized water.
RT-PCR
The SUPERSCRIP One-Step RT-PCR with PLATINUM Taq (Invitrogen Life Technologies Co.) was used to synthesize cDNA and PCR was performed as described by the manufacturer. The nucleic acid sequences of primers used for RT-PCR are shown in Table 1
. RT-PCR reactions were initially performed in a 25 µl reaction volume containing 1 µl of each primer (at 100 ng/µl) and 3 µl RNA as template. The reactions were run at 55 °C for 30 min (cDNA synthesis) and 94 °C for 2 min (predenaturation), followed by 45 cycles of 94 °C for 30 s (denaturation), 53 °C for 30 s (annealing) and 72 °C for 30 s (extension), followed by 7 min at 72 °C (final extension). To increase the detection capacity, we performed a second round of PCR. The second-round PCR reactions were carried out using Taq polymerase (Perkin-Elmer-Cetus, Norwalk, CT, USA) with 8 µl RT-PCR products as template (final 25 µl reaction mixture) under the following conditions: 35 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. In the second-round PCR, CTR was amplified using 2nd sense and anti-sense primers (Table 1). The primers of CRLR, RAMP1, 2, 3, alkaline phosphatase (ALP) and ß-actin for the second-round PCR were the same primers as those used in the initial RT-PCR. The samples were electrophoresed in 3% agarose gels and stained with ethidium bromide.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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illustrates two osteoclast-like cells before and after LCM. Two hundred and fifty cells with > three nuclei each were isolated. Total RNA was extracted and RT-PCR was used to analyze multiple gene expressions. Figure 2 shows the RT-PCR results for CTR, RAMP1, 2 and 3, CRLR and ß-actin. The predicted sizes were clearly visualized by ethidium bromide staining. RT-PCR results showed that the ubiquitous gene, ß-actin, was amplified from both spleen and osteoclast-like cells, whereas CTR mRNA was amplified from osteoclast-like cells alone. RAMP1 and RAMP3 mRNAs were amplified from spleen cells alone. RAMP2 and CRLR mRNAs were amplified from both types of cells.
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), which supported the idea that RAMP2 was amplified from osteoclast-like cells. Thus, LCM is a useful technique for isolation of small cell samples, and our strategy might be extended to other procedures, such as quantitative RT-PCR to measure mRNA levels in the osteoclast. The bone marrow macrophages are the precursors of osteoclasts; it will be interesting to compare gene expression between osteoclasts and bone marrow macrophages. Immunostaining of the Fc receptor, C3 receptor or vitamin D receptor will help to distinguish those cell types in our co-culture system. However, in order to perform RNA analysis after immunostaining, further efforts should be made to modify the conventional staining protocol to protect RNA from degradation.
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In summary, we have demonstrated that LCM is a useful solution for osteoclast research. We found that osteoclast-like cells expressed mRNAs for CTR, CRLR and RAMP2 but not RAMP1 or RAMP3; RAMP2 may therefore play an important role in osteoclast function. Further study is needed to elucidate the role of RAMP2 and its relationship to the CT family of receptors.
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Acknowledgements |
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Received 22 October 2004
Accepted 1 November 2004
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