Screening arrays to monitor apoptosis gene expression

描述（申请人提供）：一种定量高通量基因芯片 已经开发了基于核酸酶保护的测定用于药物发现。的 测定允许测量许多基因的表达水平 同时在个别样品中。我们将利用这项技术开发一种 可以测量促凋亡蛋白表达变化的测定， 用实验化合物处理的细胞中的抗凋亡基因 96孔微孔板的孔。在96孔板中进行的该测定可以是 用于筛选新的化学化合物，诱导或抑制 凋亡基因的表达。一种可以诱导 促凋亡基因和/或降低抗凋亡基因的表达 癌细胞在某些类型的癌症的治疗中可能被证明是有价值的。 我们的专利方法将核酸酶保护协议应用于基因芯片。这 允许DNA阵列技术以高通量形式使用， 灵敏度和再现性。简而言之，细胞在每个孔中生长和处理 的96孔板，在核酸酶保护存在下裂解 寡核苷酸，其与所有靶向mRNA序列杂交并保护所有靶向mRNA序列。 在核酸酶消化后，寡核苷酸化学计量地结合至靶向的寡核苷酸。 在基因芯片上对序列进行定量。该方法足够灵敏， 测量极低水平表达的基因-每个细胞低至一个mRNA分子 在30,000个细胞的样本中。

DESCRIPTION (provided by applicant): A quantitative high-throughput gene chip assay, based on nuclease protection, has been developed for drug discovery. The assay permits the expression levels of numerous genes to be measured simultaneously in individual samples. We will use this technology to develop an assay that can measure changes in the expression of pro-apoptotic and anti-apoptotic genes in cells treated with experimental compounds in a single well of a 96-well microplate. This assay, performed in 96-well plates, can be used to screen for novel chemical compounds that induce or inhibit the expression of apoptotic genes. A drug that can induce the expression of pro-apoptotic genes and/or decrease expression of anti-apoptotic genes in cancer cells may prove valuable in the treatment of some types of cancers. Our patented method applies nuclease protection protocols to gene chips. This allows DNA array technology to be used in high throughput format with both sensitivity and reproducibility. Briefly, cells, grown and treated in each well of a 96-well plate, are lysed in the presence of nuclease protection oligonucleotides, which hybridize to and protect all targeted mRNA sequences. After nuclease digestion, oligonucleotides bound stoichiometrically to targeted sequences are quantified on gene chips. The method is sensitive enough to measure genes expressed at very low levels -down to one mRNA molecule per cell in samples of 30,000 cells.