DNA in situ Hybridizations for VEGFA Gene Locus (6p12) in Human Tumor Tissue
人肿瘤组织中VEGFA基因座（6p12）的DNA原位杂交
发布: 2015年08月05日第5卷第15期 DOI: 10.21769/BioProtoc.1553 浏览次数: 9891
评审: HongLok LungMartin V KolevOmar Akil
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参见作者原研究论文
The authors used this protocol in:
Jun 2014
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Abstract
Over the last decades numerous regulators of angiogenesis have been identified and characterized. Among the others the vascular endothelial growth factor (VEGFA) appears undoubtedly important in several pathophysiological processes. Moreover, VEGFA represents one of the most attractive targets of anticancer therapy, given its major role in the growth and development of different tumor types. Here we describe a method to detect the copy number variation (CNV) status of the VEGFA gene by fluorescence in situ hybridization (FISH). FISH analysis is a reliable method for investigating VEGFA amplification or increased gene copy number and may represent an alternative method to immunohistochemical analysis for investigating the deregulation of VEGFA expression levels.
Keywords: VEGFA (VEGFA)
TMA (TMA)
FISH (鱼)
6p12 (6p12)
CNV (CNV)
Materials and Reagents
- BAC clone No. RPCIB753M0921Q (GenomeCUBE)
- LB medium (Sigma-Aldrich, catalog number: L3522-250G )
- Large-construct kit (QIAGEN, catalog number: 12462 )
- SP6 (EuroFins MGW Operon LLC A Eurofins Genomics Company, catalog number: SP050-1 ) and T7 (EuroFins MGW Operon LLC A Eurofins Genomics Company, catalog number: SP401 ) primers
- AluI restriction enzyme (Life Technologies, InvitrogenTM, catalog number: 45200-029 )
- Cy3-dUTP (GE Healthcare Dharmacon, catalog number: 45-000-738 )
- BioPrime array CGH kit (module) (Life Technologies, InvitrogenTM, catalog number: 18095-012 ) (it includes 2.5 Random primers solution, 10x dUTP nucleotide mix, Exo-Klenow fragment)
- Amicon Ultra-0.5 centrifugal filter unit with Ultracel-30 membrane (Merck KGaA, catalog number: UFC503096 )
- Human Cot-1 DNA (Life Technologies, InvitrogenTM, catalog number: 15279-011 )
- DAPI solution (1 µg/ml) (Abbott Laboratories, catalog number: 06J49-001 )
- Spectrum Green-labeled Chr6 centromeric probe (CEP6) and hybridization buffer (Abbott Laboratories, catalog number: 06J37-016 and 07J36-001 )
- Micron-centrifugal filters (Merck KGaA, catalog number: MRCF0R030 )
- TE Buffer, 1x Solution 500 ml (USB, catalog number: 75893 )
- 20x SSC (Abbott Laboratories, catalog number: 02J10-032 )
- 0.3 % NP40 (Abbott Laboratories, catalog number: 07J05-001 )
- Pretreatment reagent VP 2000 (Abbott Laboratories, catalog number: 02J06-030 )
- Protease 1 (250 mg) (Abbott Laboratories, catalog number: 02J08-032 )
- Ethanol (Sigma- Aldrich, catalog number: 0 2860 )
- Rubber cement (Carfa AG, catalog number: 00494 )
- Protease 1 (250 mg in 500 ml protease buffer) (Sauter et al., 1995)
- Wash buffer (0.4 SSC + 0.3% NP40-pH 7-7.5) (Sauter et al., 1995)
Equipment
- Incubator for slides (Memmert GmbH + Co.KG, Mode: ll IPP 300 )
- Pipette (Eppendorf)
- Nanodrop spectrophotometer assay (Thermo Fisher Scientific, model: NanoDrop 2000 )
- Slide (Thermo Fisher Scientific, catalog number: No. 10143352 )
- Cover slide (Biosystem 24 x 50 mm, catalog number: 14071135638 and R. Langenbrinck 21 x 26 mm, catalog number: 01-2126 )
- Hotplate for denaturation (Medax GmbH & Co.KG, catalog number: 12895 )
- Zeiss fluorescence microscope using a 63x objective (Carl Zeiss AG, model: Axioskop 40 )
- AxioVision software (Carl Zeiss AG, model: SE64 Rel.4.9 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Andreozzi, M., Quagliata, L., Burmeister, K., Arabi, L., Schneider, S., Tornillo, L. and Terracciano, L. (2015). DNA in situ Hybridizations for VEGFA Gene Locus (6p12) in Human Tumor Tissue. Bio-protocol 5(15): e1553. DOI: 10.21769/BioProtoc.1553.
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分类
癌症生物学 > 通用技术 > 遗传学 > 基因表达
细胞生物学 > 细胞染色 > 核酸
分子生物学 > DNA > DNA 标记
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