Chou Cheng-Chung, Chen Chun-Houh, Lee Te-Tsui, Peck Konan
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115, Republic of China.
Nucleic Acids Res. 2004 Jul 8;32(12):e99. doi: 10.1093/nar/gnh099.
Gene-specific oligonucleotide probes are currently used in microarrays to avoid cross-hybridization of highly similar sequences. We developed an approach to determine the optimal number and length of gene-specific probes for accurate transcriptional profiling studies. The study surveyed probe lengths from 25 to 1000 nt. Long probes yield better signal intensity than short probes. The signal intensity of short probes can be improved by addition of spacers or using higher probe concentration for spotting. We also found that accurate gene expression measurement can be achieved with multiple probes per gene and fewer probes are needed if longer probes rather than shorter probes are used. Based on theoretical considerations that were confirmed experimentally, our results showed that 150mer is the optimal probe length for expression measurement. Gene-specific probes can be identified using a computational approach for 150mer probes and they can be treated like long cDNA probes in terms of the hybridization reaction for high sensitivity detection. Our experimental data also show that probes which do not generate good signal intensity give erroneous expression ratio measurement results. To use microarray probes without experimental validation, gene-specific probes approximately 150mer in length are necessary. However, shorter oligonucleotide probes also work well in gene expression analysis if the probes are validated by experimental selection or if multiple probes per gene are used for expression measurement.
目前在微阵列中使用基因特异性寡核苷酸探针以避免高度相似序列的交叉杂交。我们开发了一种方法来确定用于准确转录谱研究的基因特异性探针的最佳数量和长度。该研究调查了25至1000 nt的探针长度。长探针比短探针产生更好的信号强度。短探针的信号强度可通过添加间隔物或使用更高的探针浓度进行点样来提高。我们还发现，每个基因使用多个探针可以实现准确的基因表达测量，如果使用较长的探针而不是较短的探针，则所需的探针较少。基于经实验证实的理论考虑，我们的结果表明150mer是用于表达测量的最佳探针长度。可以使用计算方法识别150mer探针的基因特异性探针，就杂交反应而言，它们可以像长cDNA探针一样用于高灵敏度检测。我们的实验数据还表明，不能产生良好信号强度的探针会给出错误的表达比率测量结果。为了在没有实验验证的情况下使用微阵列探针，长度约为150mer的基因特异性探针是必要的。然而，如果通过实验选择对探针进行验证，或者每个基因使用多个探针进行表达测量，较短的寡核苷酸探针在基因表达分析中也能很好地发挥作用。
