Boccara Martine, Sarazin Alexis, Billoud Bernard, Bulski Agnes, Chapell Louise, Baulcombe David, Colot Vincent
Unité de Recherche en Génomique Végétale, INRA/CNRS/UEVE, Evry cedex, France.
Methods Mol Biol. 2010;631:75-86. doi: 10.1007/978-1-60761-646-7_8.
Small RNA (sRNA) populations extracted from Arabidopsis plants submitted or not to biotic stress, were reverse-transcribed into cDNAs, and these were subsequently hybridized after labelling 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 small RNA populations.
从经受或未经受生物胁迫的拟南芥植株中提取的小RNA（sRNA）群体被逆转录成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，使用了八个与位于4号染色体上的序列相对应的cDNA克隆，并获得了强大而特异的杂交信号。此外，在未处理植株的情况下，沿4号染色体的杂交信号与先前通过大规模平行序列签名（MPSS）确定的sRNA丰度高度一致，但在胁迫处理后有很大差异。这些结果证明了与DNA平铺阵列杂交用于检测小RNA群体主要变化的实用性。
