Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays
Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays
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DOI：
10.1093/nar/28.22.4552
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发表时间：
2000-11-15
影响因子：
14.9
通讯作者：
Madore, SJ
中科院分区：
文献类型：
--
作者：
Kane, MD;Jatkoe, TA;Madore, SJ
为了检查 50mer 寡核苷酸（寡核苷酸探针）微阵列的实用性和性能，将基因特异性寡核苷酸探针与 PCR 探针一起点样到玻璃微阵列上，并评估每种探针类型的性能。使用具有不同程度的序列相似性的靶RNA来研究寡核苷酸探针的特异性。灵敏度被定义为检测 mRNA 3 倍变化的能力，观察到寡核苷酸探针和 PCR 探针之间的灵敏度没有显着差异，并且两者都具有类似于 10 个 mRNA 拷贝/细胞的最小可重复检测限。特异性研究表明，对于给定的寡核苷酸探针，任何“非目标”转录物 (cDNA) > 75% 相似（超过 50 个碱基目标）可能会显示交叉杂交。因此，与目标序列（在寡核苷酸探针 50 碱基目标区域内）具有 >75-80% 序列相似性的非目标序列将有助于总体信号强度。：此外，如果 50 碱基目标区域略有相似，则它不得包括一段 >15 个连续碱基的互补序列。因此，需要了解目标序列及其与目标组织或 RNA 样品中其他 mRNA 的相似性，才能设计成功的寡核苷酸探针以获得高质量的微阵列结果，这些结果共同验证了实用性：df 寡核苷酸探针 (50mer) 玻璃微阵列。
To examine the utility and performance of 50mer oligonucleotide (oligonucleotide probe) microarrays, gene-specific: oligonucleotide probes were spotted along with PCR probes onto glass microarrays and the performance of each probe type was evaluated. The specificity of oligonucleotide probes was studied using target RNAs that shared various degrees of sequence similarity. Sensitivity was defined las the ability to detect a 3-fold change in mRNA, No significant difference in sensitivity between oligonucleotide probes and PCR probes was observed and both had a minimum reproducible detection limit of similar to 10 mRNA copies/cell. Specificity studies showed that for a given oligonucleotide probe any 'non-target' transcripts (cDNAs) >75% similar-over the 50 base target may show crosshybridization. Thus non-target sequences which have >75-80% sequence similarity with target sequences (within the oligonucleotide probe 50 base target :region) will contribute to the overall signal intensity.: In addition, if the 50 base target region is marginally similar, it must not include a stretch of complementary sequence >15 contiguous bases. Therefore, knowledge about the target sequence, as well as its similarity to other mRNAs in the target tissue or RNA sample, is required to design successful oligonucleotide probes for quality microarray results, Together these results validate the utility:df oligonucleotide probe (50mer) glass microarrays.