乳腺组织染色质免疫共沉淀方法的改进

An Improved Method of Chromatin Immunoprecipitation for Mammary Gland

作为研究基因表达调控的重要方法,近年来染色质免疫共沉淀(ChIP)得到了越来越广泛的应用,但有关将乳腺组织作为ChIP材料的研究及其方法尚未见报道。采用传统的组织ChIP试剂盒处理乳腺组织时,存在样品回收率低、乳脂/乳蛋白影响杂交效率和超声积热等问题,针对以上问题对乳腺组织ChIP方法进行改进。新方法省略组织均一化步骤、用酶切-超声法代替超声法进行染色质片段化、在细胞裂解和酶切步骤间增加洗涤步骤。结果显示,与脾脏组织相比,乳腺组织不适合进行组织均一化处理。酶切-超声法比超声法更适于对乳腺组织进行染色质片段化。采用改进后的ChIP方法获得的DNA样品中,靶序列得到显著的富集,所得的DNA样品能够满足测序(ChIP-Seq)要求。结果说明,改进后的方法适用于乳腺组织染色质免疫共沉淀,并为研究乳腺组织基因表达调控奠定了基础。

Chromatin Immunoprecipitation which is an important method in gene expressing regulation has been widely used recently. However, the application of ChIP for mammary gland has not yet been reported. When the traditional tissue-ChiP kit was used in mammary gland, it had some problems, including that the low sample recovery rate, hybridization efficiency disturbed by butter fat/lactoprotein and still-accumulating heat in the sonication. In order to solve these problems, in this study, the ChIP method was improved for mammary gland. In the improved protocol, the step of homogenizing the tissue was omitted, sonication was replaced by enzyme digestion-sonication, and the washing procedure was added between swelling and enzyme digestion. The results showed that the mammary gland was not suitable for homogenizing compared with the spleen. Enzyme digestion-sonication was more suitable for shearing the chromatin than sonication. Compared with the traditional CHIP method, the target sequences were significantly enriched in the DNA sample, and the DNA sample reached the demands for sequencing by using the improved method. It indicated that the improved ChIP method was suitable for mammary gland, and it would help for the research of gene expression regulation in the mammary gland.