摘要
目的建立和优化基于全基因组DNA芯片的细菌基因表达谱技术,并用实时定量RT-PCR对此技术平台进行评价。方法提取并纯化鼠疫耶尔森菌总RNA,以六核苷酸随机引物(N6)和/或基因组特异引物(GDP)逆转录合成cDNA,用CY染料直接或间接标记后,与包括鼠疫耶尔森菌4005个ORF的全基因组芯片杂交,通过芯片扫描仪获得表达谱分析结果,归一化后进行分析。结果用实时定量PCR技术验证。结果采用N6+GDP混合引物以间接法标记获得了较为理想的结果,表达谱技术与实时定量PCR技术所得结果高度相关,证明了此技术的可靠性和实验设计与数据分析的合理性。结论合理的设计和归一化的分析方法是基于DNA芯片的细菌基因表达谱分析成功的关键,该技术的建立为细菌功能基因组研究奠定了基础。
Objective To establish and optimize the DNA microarray-based bacterial gene expression profiling, and to use the real time RT-PCR to validate the expression profiling results. Methods Total RNA extracted and purified from Y. pestis were reverse-transcribed to eDNA and labeled by CY-dye directly or indirectly with random hexa-oligonucleotide primers（N6） and genome-directed primers（GDP）. The labeled probes were hybridized to a microarray containing 4005 ORF of Y. pestis and the results were obtained by a laser scanner and analyzed by the software. RT-PCR were developed based several genes with different expression levels for confirming the expression profiling results. Results The excellent expression profiling results were obtained by using N6 ＋ GDP primers and irdirect labeling methods. The correlation between microarray-based expression profiling and real time RT-PCR was very high（ r = 0.98）, which demonstrated that the microarray-based expression profiling is a reliable technique and the experimental design and data analysis are rational. Conclusion The rational experimental design and the normalized data analysis are critical to the success of DNA microarray-based bacterial expression profiling analysis, which will lay a foundation for functional genomics studies.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2006年第2期179-184,共6页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金重点项目(30430620)
关键词
DNA芯片
细菌
基因表达谱
DNA microarray
Bacterial
Gene expression profiling
