Chip-on-beads: flow-cytometric evaluation of chromatin immunoprecipitation.
BACKGROUND
Chromatin immunoprecipitation (ChIP) is a widely used technique for the detection of in vivo DNA-protein interactions underlying epigenetic regulation. The standard readout of ChIP is based on semi-quantitative or quantitative PCR measurements; however, the development of alternative platforms with high throughput potentialities is expected to facilitate the introduction of this method into routine diagnostics.


METHODS
We have established a flow-cytometry-based alternative for the evaluation of ChIP data. The method is based on the capture of the products of a conventional PCR run to low cycle numbers, on microbeads.


RESULTS
In vivo histone H4 acetylation and H3 lysine 4 methylation was detected at the promoter of the tissue transglutaminase type 2 gene. These results were confirmed by QPCR measurements. The levels of modifications decreased significantly upon apoptosis and were accompanied by the down-regulation of TGM2 mRNA expression.


CONCLUSIONS
This method that we named ChIP-on-beads, a combination of flow cytometry and conventional PCR, is a reliable and efficient alternative in the quantitative analysis of ChIP results, especially promising when high throughput monitoring of epigenetic markers of diagnostic importance is required. The method is simple enough to be easily implemented in a routine flow-cytometric laboratory.
Székvölgyi
Lóránt
L
Department of Biophysics and Cell Biology, University Medical School of Debrecen 4012 Debrecen, Hungary.
Bálint
Bálint L
BL
Imre
László
L
Goda
Katalin
K
Szabó
Miklós
M
Nagy
László
L
Szabó
Gábor
G
D000107:Acetylation; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D015536:Down-Regulation; D005434:Flow Cytometry; D019204:GTP-Binding Proteins; D006657:Histones / Q000378:metabolism; D008745:Methylation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012333:RNA, Messenger / Q000096:biosynthesis; D011503:Transglutaminases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D006657:Histones; D012333:RNA, Messenger; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins