Methyl DNA Immunoprecipitation

互联网2013-09-06

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Epigenetics is the study of heritable changes in gene expression. Chromatin immunoprecipitation (ChIP) and methylation status analysis of genes have been applied to the study of epigenetic modifications, often perturbed in human cancer. ChIP is a technique allowing the analysis of the protein association with specific genomic regions in the context of intact cells. ChIP and immunoprecipitation (IP) of methylated DNA, both rely on the use of well-characterized specific antibodies. The first is described in Chapter 2 and the second is shown here. At Diagenode, a novel METHYL kit has been designed to immunoprecipitate methylated DNA (Methyl DNA IP). This kit allows you to perform DNA methylation analysis of your sample together with optimized internal IP controls, all in one tube. This brand new Methyl DNA IP method provides methylated DNA (meDNA) and unmethylated DNA (unDNA) controls to be used together with your DNA sample, allowing direct correlation between immunoprecipitated material and methylation status. Such methylation analysis is highly specific and each IP is quality controlled, two essential keys for reliable results. In addition, the kit protocol is fast and user-friendly.

Key Words: Immunoprecipitation - methylated DNA - MeDIP - internal controls

15.1 Introduction

Current methods used for the detection of methylated DNA are methods based on methylation-sensitive enzymatic digestion (1 ), bisulfite treatment (2 �4 ), enrichment by binding to methylated binding domain (MBD) (5 �8 ), and enrichment by immunoprecipitation of methylated DNA (9 ). DNA methylation detection assays using methylation-sensitive restriction enzymes to digest unmethylated DNA while leaving methylated DNA intact are DNA-sequence dependent and not compatible with high-throughput (HTP) analysis. The use of sodium bisulfite to deaminate cytosine to uracil while leaving 5-methylcytosine intact is a cumbersome technique, which involves subsequent optimization of specific PCR or sequencing, involving time- and labor-intensive chemical treatments that damage DNA and limit throughput. It was shown that the methyl-binding domain (MBD) of MeCP2 has some sequence preference besides its recognition of methyl-CpGs (10 ) and that the MBD method does require relatively high methyl-CpG density (11 ). Methylated DNA enrichment by binding to MBDs can, therefore, be sequence dependent and can also show low specificity and potential for false-positive results due to capture of unmethylated DNA. The MBD method combined with enzymatic restriction of the DNA has been adapted to HTP (8 , 12 ).

Yet another way to enrich for methylated DNA is by Methyl DNA IP. Methyl DNA IP uses bead-immobilized anti-5-methyl cytosine antibodies to isolate the methylated DNA, which allows highly efficient enrichment of methylated DNA dose-dependent and sequence-independent, with high specificity. Moreover, the use of the Methyl DNA IP technique is compatible with high-throughput platforms (13 ) and can directly give reliable qualitative results as well as semi-quantitative data. The use of antibody instead of MBD to pull-down methylated sequences presents, therefore, several advantages. In addition, it is important to point out that monoclonal antibody is produced with far less lot-to-lot variation in comparison with a fusion protein expressed in E. coli.

Our novel METHYL kit is designed to immunoprecipitate methylated DNA. This kit allows you to perform DNA methylation analysis of your sample including optimized internal IP controls. The internal IP controls consist of methylated DNA (meDNA) and unmethylated DNA (unDNA) added to your DNA sample, such that a direct correlation between immunoprecipitated material and methylation status can be done. This methylation analysis is highly specific due to the use of a well-characterized monoclonal antibody and each IP is directly quality controlled: two essential keys for reliable results. In addition, the kit protocol is fast and user-friendly. The METHYL kit includes three modules; they are used sequentially as follows for genomic DNA preparation (see Section 3.1), immunoprecipitation of methylated DNA (see Section 3.2), and qPCR analysis of the immunoprecipitated DNA (see Section 3.3 and Table 15.1 ). Each module is provided with adapted buffers and detailed protocols.

Table 15.1 qPCR module following Methyl DNA IP

Primer pairs (10 μM each)	Specificity (size of amplified DNA)	Input DNA sample (which includes Ctrls) amplification:	Methyl DNA IP (which includes Ctrls) amplification:
hum meDNA primer pair (AlphaX1)	Human DNA (81 bp)	Yes (if sample is )	Yes
hum unDNA primer pair (GAPDH)	Human DNA (102 bp)	Yes (human DNA )	No
meDNA pos control primer pair #1	Kit Positive Ctrl (81 bp)	Yes	Yes
meDNA pos control primer pair #2	Kit Positive Ctrl (87 bp)	Yes	Yes
unDNA neg control primer pair #1	Kit Negative Ctrl (84 bp)	Yes	No
unDNA neg control primer pair #2	Kit Negative Ctrl (92 bp)	Yes	No

15.2 Materials

15.2.1 DNA Preparation and Shearing

1.	Cultured cells and Trypsin�EDTA.
2.	GenDNA module (cat. no. mc-green-002, Diagenode).
3.	Phosphate buffered saline (PBS).
4.	Phenol:chloroform:isoamyl alcohol (25:24:1), chloroform:isoamyl alcohol (24:1), 100% ethanol, 70% ethanol. Fume hood. Vortex.
5.	Agarose and TAE buffer, DNA molecular weight marker.
6.	Bioruptor (cat. no. UCD-200, Diagenode).

15.2.2 Methylated DNA Immunoprecipitation and Analysis of Immunoprecipitated DNA

1.	METHYL kit (cat. no. mc-green-003, Diagenode), which includes the Methyl DNA IP module (cat. no. mc-green-001) and the GenDNA and qPCR modules (cat. no. mc-green-002).
2.	Autoclaved tips.
3.	Rotating wheel.
4.	Thermomixer (50 and 65°C).
5.	Incubator (37°C).
6.	Quantitative PCR facilities and reagents.

15.3 Methods

15.3.1 DNA Preparation and Module

The GenDNA module from Diagenode has been optimized for the preparation of genomic DNA from cultured cells to be then used in Methyl DNA IP (see Note 1 ). The goal of this first step is to get high molecular weight genomic DNA. Cell culturing is the starting point before cell collection and lysis described below.

15.3.1.1 Cell Collection and Lysis

1.	Pellet suspension culture out of its serum-containing medium. Trypsinize adherent cells and collect cells from the flask. Centrifuge at 300g for 5 min at 4°C.
2.	Discard the supernatant. Resuspend cells in 5�10 mL ice-cold PBS. Centrifuge at 500g for 5 min. Discard the supernatant. Repeat this resuspension and centrifugation step once more. This step is to wash the cells.
3.	Meanwhile, place the GenDNA digestion buffer at room temperature (RT) and the GenDNA proteinase K on ice.
4.	Add GenDNA proteinase K to the GenDNA digestion buffer before use. The stock of provided proteinase K is 200X; e.g., add 5 µL per 1 mL of digestion buffer, i.e., the freshly prepared complete digestion buffer to be used directly.
5.	Resuspend cells in complete digestion buffer (1 volume). For 3 million cells, use 300 µL complete digestion buffer. For 10 million cells, use 500 µL complete digestion buffer. It might be necessary to use more buffer to avoid problems when performing the extractions below. If necessary, for 3 million cells, use up to 600 µL of buffer. For 10 million cells, use up to 1,000 µL of buffer.
6.	Cell lysis: Incubate the samples with shaking at 50°C for 12�18 h in tightly capped tubes. That is the cell lysis step. At this stage, samples are viscous. After 12 h incubation the tissue should be almost indiscernible, a sludge should be apparent from the organ samples, and tissue culture cells should be relatively clear.

15.3.1.2 Extraction of Nucleic Acids and DNA Purification

1.	Thoroughly extract the samples with an equal volume of phenol:chloroform:isoamyl alcohol. Add 1 volume of phenol:chloroform:isoamyl alcohol (25:24:1). One volume is about 500 µL. It is possible to incubate the samples at RT for 10 min on a rotating wheel before centrifugation. Use gentle rotation and do not vortex. Work under a fume hood.
2.	Centrifuge at 1,700g for 10 min in a swinging bucket rotor.
3.	Transfer the aqueous (top) layer to a new tube. Increase volume if necessary (see above) and pipette slowly.
4.	Add 1/2 volume of GenDNA precipitant and 2 volumes of 100% ethanol (see Note 2 ). That is to purify the DNA. One volume is about 500 µL and corresponds to the original amount of top layer. Add therefore 250 µL of precipitant and 1,000 µL of 100% ethanol. The DNA should immediately form a stringy precipitate.
5.	Recover DNA by centrifugation at 1,700g for 2 min. This brief precipitation in the presence of an optimized high salt precipitant (GenDNA precipitant) reduces the amount of RNA in the DNA sample. For long-term storage, it is convenient to leave the DNA in the presence of ethanol.
6.	Rinse the pellet with 70% ethanol. Decant ethanol and air-dry the pellet. It is important to rinse extensively to remove any residual of salt and phenol.
7.	Resuspend the pellet of DNA at ∼1 mg/mL in GenDNA TE until dissolved. Shake gently at room temperature or at 65°C for several hours to facilitate solubilization. Store at 4°C. From 3 million cells, ∼20�30 µg of DNA can be expected in a volume of 20�30 µL. From 10 million cells, ∼50�100 µg of DNA can be expected (in a volume of 200�300 µL). If possible, it is recommended to get at least 30 µg of DNA (when enough material is available) to be able to work with 30 µg of DNA (see Section 3.1.3).
8.	If necessary, residual RNA can be removed at this step by adding 2 µL of GenDNA RNase (DNase-free) per milliliter of DNA sample and incubating 1 h at 37°C, followed by phenol:chloroform extraction and ethanol precipitation (similar to above).
9.	For DNA analysis, run samples in a 1% agarose gel along with DNA size marker to visualize the DNA preparation efficiency.

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