|
|
|
| An Improved Method of Chromatin Immunoprecipitation for Mammary Gland |
| REN Yan-ping1,2, LUO Chan1, HUANG Zhang-hu2, HUANG Shi-hai3, SHI De-shun1, LI Xiang-ping1 |
1. State Key Laboratory of Subtropical Bioresource Conservation and Utilization, Guangxi University, Guangxi 530005, China;
2. School of Basic Medical Sciences, Zunyi Medical University, Zunyi 563003, China;
3. College of Life Science and Technology, Guangxi University, Nanning 530005, China |
|
|
|
Abstract 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.
|
|
Received: 30 June 2014
Published: 25 September 2014
|
|
|
|
|
|
[1] Rosenau C, Emery D, Kaboord B, et al. Development of a high-throughput plate-based chemiluminescent transcription factor assay. Journal of Biomolecular Screening, 2004,9(4): 334-342.
[2] Shao W, Wei H J, Qiao J Y, et al. Parallel profiling of active transcription factors using an oligonucleotide array-based transcription factor assay (OATFA). Journal of Proteome Research, 2005, 4(4): 1451-1456.
[3] Hellman L M,Fried M G. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nature Protocols, 2007, 2(8): 1849-1861.
[4] Fried M G. Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis, 1989, 10(5-6): 366-376.
[5] Feng S Y, Ota K, Yamada Y, et al. A yeast one-hybrid system to detect methylation-dependent DNA-protein interactions. Biochemical and Biophysical Research Communications, 2004, 313(4): 922-925.
[6] Philp J A, Burdon T G, Watson C J. Differential activation of STATs 3 and 5 during mammary gland development. FEBS Letters, 1996, 396(1): 77-80.
[7] Orlando V, Strutt H, Paro R. Analysis of Chromatin Structure by in Vivo Formaldehyde Cross-Linking. Methods, 1997, 11(2): 205-214.
[8] Orlando V,Paro R. Mapping Polycomb-repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin. Cell, 1993, 75(6): 1187-1198.
[9] Jackson V. Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent. Cell, 1978, 15(3): 945-954.
[10] Robertson G, Hirst M, Bainbridge M, et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nature Methods, 2007, 4(8): 651-657.
[11] Weinmann A S, Bartley S M, Zhang T, et al. Use of chromatin immunoprecipitation to clone novel E2F target promoters. Molecular and Cellular Biology, 2001, 21(20): 6820-6832.
[12] Oh S W, Mukhopadhyay A, Dixit B L, et al. Identification of direct DAF-16 targets controlling longevity, metabolism and diapause by chromatin immunoprecipitation. Nature genetics, 2006, 38(2): 251-257.
[13] Filant J, Lydon J P, Spencer T E. Integrated chromatin immunoprecipitation sequencing and microarray analysis identifies FOXA2 target genes in the glands of the mouse uterus. The FASEB Journal, 2014, 28(1): 230-243.
[14] Kawazu M, Saso K, Tong K I, et al. Histone demethylase JMJD2B functions as a co-factor of estrogen receptor in breast cancer proliferation and mammary gland development. PLoS One, 2011, 6(3): e17830.
[15] Mazumdar A, Wang R-A, Mishra S K, et al. Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor. Nature Cell Biology, 2001, 3(1): 30-37.
[16] Kang S-H L, Vieira K, Bungert J. Combining chromatin immunoprecipitation and DNA footprinting: a novel method to analyze protein-DNA interactions in vivo. Nucleic acids Research, 2002, 30(10): e44-e44.
[17] Zhou Z J, Dai Z, Zhou S L, et al. HNRNPAB Induces Epithelial-Mesenchymal Transition and Promotes Metastasis of Hepatocellular Carcinoma by Transcriptionally Activating SNAIL. Cancer Research, 2014, 74(10): 2750-2762.
[18] Dahl J A,Collas P. A rapid micro chromatin immunoprecipitation assay (ChIP). Nature Protocols, 2008, 3(6): 1032-1045.
[19] Fanelli M, Amatori S, Barozzi I, et al. Chromatin immunoprecipitation and high-throughput sequencing from paraffin-embedded pathology tissue. Nature Protocols, 2011, 6(12): 1905-1919.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
