Martel Ralph R, Botros Ihab W, Rounseville Matthew P, Hinton James P, Staples Robin R, Morales David A, Farmer John B, Seligmann Bruce E
High Throughput Genomics, Inc, Tucson, AZ 85712, USA.
Assay Drug Dev Technol. 2002 Nov;1(1 Pt 1):61-71. doi: 10.1089/154065802761001310.
The principles and performance are described for the ArrayPlate mRNA assay, a multiplexed mRNA assay for high-throughput and high-content screening and drug development. THP-1 monocytes grown and subjected to compound treatments in 96-well plates were subjected to a multiplexed nuclease protection assay in situ. The nuclease protection assay destroyed all cell-derived mRNA, but left intact stoichiometric amounts of 16 target-specific oligonucleotide probes. Upon transfer of processed cell lysates to a microplate that contained a 16-element oligonucleotide array at the bottom of each well, the various probe species were separated by immobilization at predefined elements of the array. Quantitative detection of array-bound probes was by enzyme-mediated chemiluminescence. A high-resolution charge-coupled device imager was used for the simultaneous readout of all 1536 array elements in a 96-well plate. For the measurement of 16 genes in samples of 25000 cells, the average standard deviation from well to well within a plate was 8.6% of signal intensity and was 10.8% from plate to plate. Assay response was linear and reproducibility was constant for all detected genes in samples ranging from 1000 to 50000 cells. When THP-1 monocytes were differentiated with phorbol ester and subsequently activated with bacterial lipopolysaccharide that contained different concentrations of dexamethasone, dose-dependent effects of dexamethasone on the mRNA levels of several genes were observed.
本文描述了ArrayPlate mRNA检测法的原理和性能，这是一种用于高通量、高内涵筛选及药物研发的多重mRNA检测法。在96孔板中培养并接受化合物处理的THP-1单核细胞，接受了原位多重核酸酶保护检测。核酸酶保护检测破坏了所有细胞来源的mRNA，但使化学计量的16种靶标特异性寡核苷酸探针保持完整。将处理后的细胞裂解物转移至每个孔底部含有16元素寡核苷酸阵列的微孔板后，各种探针通过固定在阵列的预定义元件上而分离。通过酶介导的化学发光对结合在阵列上的探针进行定量检测。使用高分辨率电荷耦合器件成像仪同时读取96孔板中的所有1536个阵列元件。对于25000个细胞样本中16个基因的测量，同一板内各孔之间信号强度的平均标准差为8.6%，板间为10.8%。在1000至50000个细胞的样本中，所有检测基因的检测响应呈线性，重复性良好。当用佛波酯诱导THP-1单核细胞分化，随后用含有不同浓度地塞米松的细菌脂多糖激活时，观察到地塞米松对几个基因mRNA水平的剂量依赖性效应。
