Kenzelmann Marc, Klären Ralf, Hergenhahn Manfred, Bonrouhi Mahnaz, Gröne Hermann-Josef, Schmid Wolfgang, Schütz Günther
Division of Molecular Biology of the Cell I, Deutsches Krebsforschungszentrum Heidelberg, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
Genomics. 2004 Apr;83(4):550-8. doi: 10.1016/j.ygeno.2003.09.026.
Microarray-based gene profiling of laser-assisted microdissected tissues or clinical biopsies is still a challenge since the amount of total RNA in such samples is limited and amplification of RNA is mandatory. Representative amplification of mRNA is highly dependent on the reverse transcription reaction, which is error prone, and on the number of amplification cycles. To improve the accuracy of RNA amplification, we optimized, combined, and tested different amplification strategies for Affymetrix oligonucleotide array hybridization. We demonstrate that different protocols differ significantly in quality of mRNA amplification. To demonstrate the accuracy and reproducibility of our optimized protocol in a clinical setting, we analyzed total RNAs from laser-assisted, microdissected cells of human prostate tissues. On the basis of these results, we recommend a standard reverse transcription reaction for small-sample-transcriptome profiling experiments as part of the Minimal Information about a Microarray Experiment (MIAME) set of standards.
基于微阵列的激光辅助显微切割组织或临床活检样本的基因谱分析仍然是一项挑战，因为此类样本中的总RNA量有限，RNA扩增必不可少。mRNA的代表性扩增高度依赖于易出错的逆转录反应以及扩增循环次数。为提高RNA扩增的准确性，我们针对Affymetrix寡核苷酸阵列杂交优化、组合并测试了不同的扩增策略。我们证明不同方案在mRNA扩增质量上存在显著差异。为在临床环境中证明我们优化方案的准确性和可重复性，我们分析了来自人前列腺组织激光辅助显微切割细胞的总RNA。基于这些结果，作为微阵列实验最少信息（MIAME）标准集的一部分，我们推荐在小样本转录组谱分析实验中采用标准逆转录反应。
