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Volume 55 (2011)
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Volume 55, Issue 1
Displaying 1-4 of 4 articles from this issue
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Mayu Takeda, Tadao Funato, Schinichi Fujimaki, Yoshinori Iwatani2011 Volume 55 Issue 1 Pages 1-4
Published: 2011
Released on J-STAGE: June 22, 2011
JOURNAL FREE ACCESSRNA degradation is a critical problem in all experimental settings and for clinical samples for the analysis of gene expression. The sufficient integrity of starting RNA is the essential factor that determines the accuracy of mRNA measurements such as quantitative real-time reverse-transcription PCR (RT-qPCR) and microarray experiments. We evaluated RNA yield and quality of RNA samples and obtained RNA Integrity Number (RIN) with a microchip electrophoresis system. All samples of frozen RNA and RNA extracted from cultured cells revealed RIN of more than 9.8, and showed a discrete, high-molecular-weight band on agarose gel electrophoresis. RIN was not affected by repeated freezing and thawing. These results suggested that the RIN could be a suitable indicator for the integrity of RNA during storage.
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Noriaki Arakawa, Yusuke Masuishi, Hisashi Hirano2011 Volume 55 Issue 1 Pages 5-8
Published: 2011
Released on J-STAGE: July 14, 2011
JOURNAL FREE ACCESSProteomics is a useful aid to discover new diagnosis markers or drug-targets for various diseases. To identify proteins expressed specifically in ovarian clear cell adenocarcinoma (CCA), a malignant strain of ovarian cancer subtype, we compared the proteomic patterns of cell lines derived from CCA and non-CCA using Two-Dimensional Differential Gel Electrophoresis (2D-DIGE) analysis. We identified several proteins, including annexin IV, which were increased in several CCA cell lines. These proteins were verified to be upregulated in several CCA cell lines by Western blot and quantitative RT-PCR analyses. Promoter analysis of annexin IV gene revealed that the p53-binding motif was involved in CCA-specific expression. Mutations in the p53 gene were absent in several CCA-derived cell lines, and gene silencing of p53 by RNA interference decreased the ANX4 expression in the CCA cell lines. Thus, the annexin IV gene is regulated by p53 in the CCA cells. Elevations of several p53-induced proteins were also observed in CCA cells. Therefore, we concluded that p53 functional status is involved in forming the unique features of the proteome in CCA cell lines.
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Takafumi Fukui, Hideaki Maruse, Arata Takahashi, Yosuke Furui, Nobuhis ...2011 Volume 55 Issue 1 Pages 9-11
Published: 2011
Released on J-STAGE: July 14, 2011
JOURNAL FREE ACCESSMLPA was developed by Schouten JP et al. in 2002, and has become a rapidly growing technique used in clinical diagnosis for genetic diseases, such as Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD), Lynch syndrome and multiple congenital anomalies (MCA)/mental retardation (MR). MLPA is a simple and rapid method for simultaneous quantification of up to 40 nucleic acid sequences in a single reaction. MLPA probe consists of two different oligonucleotides, each containing one of the PCR primer sequences as well as a sequence complementary to the target sequence. The two probe oligonucleotides hybridize to immediately adjacent target sequences. It is only when the two probe parts are both hybridized to their target sequence that they can be ligated to each other by a thermostable ligase. These ligated probes will be amplified exponentially during PCR reaction. The resulting PCR amplification products are subsequently separated by capillary gel electrophoresis. The number of probe ligation products of one probe depends on the number of target sequences in the sample. The DMD gene is located on Xp21 and the gene responsible for DMD/BMD. Large deletions of DMD gene are found in 60%, large duplications are 10%, and small mutations are 30% of the cases. Although the conventional multiplex PCR assay was developed for the genetic testing of the DMD gene, it could not detect large duplication because of its low quantitative performance. The MLPA assay can provide a simple and rapid method for the large deletion and duplication screening done in DMD/BMD patients.
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Tadashi Kondo2011 Volume 55 Issue 1 Pages 13-16
Published: 2011
Released on J-STAGE: November 16, 2011
JOURNAL FREE ACCESSCancer is a genetically and clinically diverse disease, and patients with cancers of similar clinical stages often exhibit different responses to treatment. To develop optimized therapeutic strategies, improved characterization of cancer phenotypes has long been desired. Electrophoresis is a fundamental technique to separate and quantify various molecules, and it allows molecular profiling of tumor tissues and body fluids. By combining the molecular profiles of electrophoresis and clinicopathological data, it is possible to identify key molecules that regulate malignant features and influence clinical outcomes. Such molecules should be considered as biomarker candidates for the characterization of cancer cells. Since these molecules are likely to be involved in the molecular pathways for metastasis, recurrence, and resistance to treatments, they can also be used as therapeutic targets. With an aim to develop biomarkers for characterizing cancer phenotypes, we created global protein expression profiles by electrophoresis and attempted to identify proteins that influenced clinical outcomes. A novel electrophoresis method such as two-dimensional difference gel electrophoresis (2D-DIGE) enables quantitative, reproducible, sensitive, high throughput, and exhaustive global protein expression profiling. Using 2D-DIGE and a large scale clinical sample set with a detailed clinicopathological data set, we identified proteins that influenced metastasis after surgery and induced resistance to standard chemotherapy. Another approach to identify these proteins is by using antibodies, wherein protein expression profiles are created by western blotting using a large-scale antibody library. Hence, we conclude that electrophoresis is a powerful tool for developing cancer biomarkers.
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