Goodsaid Federico M, Smith Roger J, Rosenblum I Y
Division of Drug Safety and Metabolism, Schering-Plough Research Institute, 144 Route 94, PO Box 32, Lafayette, NJ 07848, USA.
Environ Health Perspect. 2004 Mar;112(4):456-60. doi: 10.1289/ehp.6695.
Differences in hybridization platforms used in gene array analysis experiments can lead to significant differences in hybridization results. In this study we used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to investigate discrepant results between the National Institute of Environmental Health Sciences cDNA and Affymetrix oligo platforms used to evaluate hepatic gene expression changes in rats exposed to methapyrilene. Caldesmon cDNA platform hybridization results showed decreases in gene expression levels for the high-dose methapyrilene 7-day pooled samples compared with their controls. By contrast, the Affymetrix oligonucleotide platform showed increases in expression levels for these samples. Quantitative gene expression measurements provide an explanation for the discrepancies observed for these samples. In the case of caldesmon, there is a 74-base sequence in the cDNA clone that is absent in the Affymetrix sequence. The amplicon based on the cDNA clone shows > 100-fold suppression relative to the day 7 high-dose methapyrilene-pooled control. These data demonstrate the importance of using a "gold standard," such as qRT-PCR to confirm key hybridization results as well as to understand the sources of discrepancies resulting from different hybridization platforms.
基因阵列分析实验中使用的杂交平台差异可能导致杂交结果出现显著差异。在本研究中，我们使用定量逆转录-聚合酶链反应（qRT-PCR）来研究美国国家环境卫生科学研究所的cDNA平台与用于评估暴露于甲吡咯烷的大鼠肝脏基因表达变化的Affymetrix寡核苷酸平台之间的结果差异。钙调蛋白cDNA平台杂交结果显示，与对照组相比，高剂量甲吡咯烷7天混合样本的基因表达水平降低。相比之下，Affymetrix寡核苷酸平台显示这些样本的表达水平增加。定量基因表达测量为这些样本中观察到的差异提供了解释。就钙调蛋白而言，cDNA克隆中有一个74个碱基的序列在Affymetrix序列中不存在。基于cDNA克隆的扩增子相对于第7天高剂量甲吡咯烷混合对照显示出>100倍的抑制。这些数据证明了使用“金标准”（如qRT-PCR）来确认关键杂交结果以及了解不同杂交平台产生差异的来源的重要性。
