Ivanov Sergey V, Ivanova Alla V
SAIC-Frederick, NCI Center for Cancer Research, Frederick, MD, USA.
Methods Mol Biol. 2006;317:287-98. doi: 10.1385/1-59259-968-0:287.
Efficient handling of multiple reactions is a crucial prerequisite for productive RNA differential display (DD) analysis. To identify transcriptional targets of the histone H3 Lys9-specific methyltransferase Clr4, we applied a multiformat modification of DD to compare between clr4+ and clr4- transcriptomes of Schizosaccaromyces pombe. As a result, 14 differentially expressed bands were identified among 720 polymerase chain reaction (PCR) studied. The content of these bands was then analyzed by cloning, sequencing, and Northern analysis. In the final stage of verification, four Clr4 targets were isolated based on their expression in six Clr4 chromo and SET domain mutant strains. The step-by-step description of the multiformat DD provided below includes RNA purification, cDNA synthesis, 96-well PCR, electrophoretic separation of PCR products, isolation of DNA fragments from differentially expressed bands, and verification of candidate genes by Northern analysis.
高效处理多个反应是进行高效RNA差异显示（DD）分析的关键前提。为了鉴定组蛋白H3赖氨酸9特异性甲基转移酶Clr4的转录靶点，我们对DD进行了多种形式的改进，以比较粟酒裂殖酵母clr4 +和clr4 -转录组。结果，在研究的720个聚合酶链反应（PCR）中鉴定出14条差异表达条带。然后通过克隆、测序和Northern分析对这些条带的内容进行分析。在验证的最后阶段，根据四个Clr4靶点在六个Clr4染色体和SET结构域突变菌株中的表达情况将其分离出来。下面提供的多种形式DD的分步描述包括RNA纯化、cDNA合成、96孔PCR、PCR产物的电泳分离、从差异表达条带中分离DNA片段以及通过Northern分析验证候选基因。
