Laser capture microdissection-generated target sample for high-density oligonucleotide array hybridization
Laser capture microdissection-generated target sample for high-density oligonucleotide array hybridization
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DOI：
10.2144/00293st05
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发表时间：
2000-09-01
期刊：
影响因子：
2.7
通讯作者：
Todd, R
中科院分区：
文献类型：
--
作者：
Ohyama, H;Zhang, X;Todd, R
目前生物分子技术的进步允许对负责人类疾病发病机制的特定细胞进行精确的遗传指纹分析。本研究证明了从激光捕获显微切割（LCM）组织中产生靶样品的可行性，该靶样品适合用于基因表达谱的高密度寡核苷酸阵列杂交。通过LCM成功地从三对卵圆癌标本中分离出RNA，并使用T7 RNA聚合酶进行线性扩增。cDNA的评估显示，检测到五种细胞维持转录物中的五种。产生生物素化的cRNA并与人测试I基因芯片探针阵列杂交，随后将样品与HuGenFL基因芯片探针阵列杂交，显示26.5%-33.0%的RNA被检测到。大约7000个代表性基因中的每一个在六个样品中表达。这些结果表明，LCM产生的组织可以产生足够质量的cRNA用于高密度寡核苷酸微阵列分析，这是使用这种高通量技术确定全面基因表达谱的重要步骤。
Current advances in biomolecular technology allow precise genetic fingerprinting of specific cells responsible for the pathogenesis of human diseases. This study demonstrates the feasibility of generating target samples from laser capture microdissection (LCM) tissues suitable for hybridization of high-density oligonucleotide arrays for gene expression profiling. RNA was successfully isolated by LCM from three paired specimens of oval cancer and linearly amplified using T7 RNA polymerase. Evaluation of the cDNA revealed that five of five cellular maintenance transcripts are detected Biotinylated cRNA was generated and hybridized to the human Test I GeneChip(R) probe arrays, which demonstrated that the RNA is of sufficient quality and integrity to warrant further analysis Subsequent hybridization of the samples to the HuGenFL GeneChip probe arrays revealed that 26.5%-33.0% of the approximately 7000 represented genes are expressed in each of the six samples. These results demonstrate that LCM-generated tissues can generate sufficient quality cRNA for high-density oligonucleotide microarray analysis, an important step in determining comprehensive gene expression profiling using this high-throughput technology.