Boccara Martine, Sarazin Alexis, Billoud Bernard, Bulski Agnes, Chapell Louise, Baulcombe David, Colot Vincent
PSL Research University, Institut de Biologie de l'Ecole Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U1024, Ecole NormaleSupérieure, 46 rue d'Ulm, Paris, F75005, France.
Atelier de bioinformatique, Sorbonne Universités, UMR 7205 (MNHN, UPMC, CNRS, EPHE), Museum national d'histoire naturelle, rueBuffon, Paris, F-7523, France.
Methods Mol Biol. 2017;1456:127-139. doi: 10.1007/978-1-4899-7708-3_11.
Epigenetic response to stress in plants involves changes in DNA methylation, histone modifications, and expression of small noncoding RNAs (sRNA). Here we present the method of analysis of differential expression of sRNA populations using DNA tiling arrays. sRNA extracted from Arabidopsis thaliana plants exposed to pathogen elicitor or control plants were reverse-transcribed into cDNAs, and subsequently hybridized after labeling to a custom-made DNA tiling array covering Arabidopsis chromosome 4. We first designed a control experiment with eight cDNA clones corresponding to sequences located on chromosome 4 and obtained robust and specific hybridization signals. Furthermore, hybridization signals along chromosome 4 were in good agreement with sRNA abundance as previously determined by massive parallel sequence signature (MPSS) in the case of untreated plants, but differed substantially after stress treatment. These results demonstrate the utility of hybridization to DNA tiling arrays to detect major changes in sRNA abundance.
植物对胁迫的表观遗传反应涉及DNA甲基化、组蛋白修饰以及小非编码RNA（sRNA）的表达变化。本文我们介绍了使用DNA平铺阵列分析sRNA群体差异表达的方法。从暴露于病原体激发子的拟南芥植物或对照植物中提取的sRNA被逆转录成cDNA，随后在标记后与覆盖拟南芥4号染色体的定制DNA平铺阵列杂交。我们首先设计了一个对照实验，使用八个与位于4号染色体上的序列相对应的cDNA克隆，并获得了稳健且特异的杂交信号。此外，在未处理植物的情况下，沿4号染色体的杂交信号与先前通过大规模平行序列标签（MPSS）确定的sRNA丰度高度一致，但在胁迫处理后有显著差异。这些结果证明了与DNA平铺阵列杂交用于检测sRNA丰度主要变化的实用性。
