Sterrenburg Ellen, Turk Rolf, Boer Judith M, van Ommen Gertjan B, den Dunnen Johan T
Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, 2333AL Leiden, Nederland.
Nucleic Acids Res. 2002 Nov 1;30(21):e116. doi: 10.1093/nar/gnf115.
Comparisons of expression levels across different cDNA microarray experiments are easier when a common reference is co-hybridized to every microarray. Often this reference consists of one experimental control sample, a pool of cell lines or a mix of all samples to be analyzed. We have developed an alternative common reference consisting of a mix of the products that are spotted on the array. Pooling part of the cDNA PCR products before they are printed and their subsequent amplification towards either sense or antisense cRNA provides an excellent common reference. Our results show that this reference yields a reproducible hybridization signal in 99.5% of the cDNA probes spotted on the array. Accordingly, a ratio can be calculated for every spot, and expression levels across different hybridizations can be compared. In dye-swap experiments this reference shows no significant ratio differences, with 95% of the spots within an interval of +/-0.2-fold change. The described method can be used in hybridizations with both amplified and non-amplified targets, is time saving and provides a constant batch of common reference that lasts for thousands of hybridizations.
当一个共同的参照样本与每块微阵列共同杂交时，不同cDNA微阵列实验间的表达水平比较会更容易。通常这个参照样本由一个实验对照样本、一组细胞系或所有待分析样本的混合物组成。我们开发了一种替代的共同参照样本，它由点样在阵列上的产物混合物组成。在cDNA PCR产物打印之前将其部分混合，并随后向正义或反义cRNA进行扩增，可提供一个出色的共同参照样本。我们的结果表明，该参照样本在阵列上99.5%的cDNA探针中产生了可重复的杂交信号。因此，可以为每个点计算一个比率，并比较不同杂交间的表达水平。在染料交换实验中，该参照样本未显示出显著的比率差异，95%的点在+/-0.2倍变化的区间内。所描述的方法可用于扩增和未扩增靶标的杂交，节省时间，并提供一批持续数千次杂交的恒定共同参照样本。
