Support | FAQs | MeDIP

Frequently Asked Questions


Sample types and amounts for the MeDIP kit


Cell lysis and DNA fragmentation


Bead, Antibody and Immunoprecipitation - related questions


Internal Control - related questions


DNA purification - related questions


PCR & PCR Control - related questions


DNA quality, characteristics and enrichment after immunoprecipitation


Alternative uses / differences from other protocols for the MeDIP kit


Amplification


Downstream applications: microarrays, sequencing - related questions


Sample types and amount of DNA


gDNA preparation for immunoprecipitation


Immunoprecipitation - related questions


DNA elution and purification


Control - related questions


PCR - related questions


Amplification


Downstream applications: microarray, sequencing


Sample types and amounts for the MeDIP kit

Can the Diagenode MeDIP kits be used with DNA obtained from paraffin-embedded samples?

Diagenode has not tested its MeDIP kits for immunoprecipitating DNA from paraffin-embedded samples. It should be possible to perform MeDIP but note, that the quality of DNA from paraffin-embedded samples is not optimal for MeDIP. The fixation introduces DNA modifications (addition of mono-methyl group, formation of bridges between adjacent bases, generation of apurinic and apyrimidinic sites and strand breaks). DNA quality varies from sample-to sample.

However, our customers have successfully used the antibody anty-5methyl cytidine (the same as used in our MeDIP kits) in immonohistochemistry assay on fixed sections.

Is the MeDIP kit suitable for comparing DNA methylation of bacterial DNA?

Our MeDIP kit can be used for the DNA methylation studies in different organisms. In the kit we use the antibody raised against 5-methyl-cytosine. In bacteria the methylation happens on cytosine and adenosine residue; so our kit can be used only for cytosine methylation studies.

If you decide to use this kit (so if the methylation concern the cytosine) you can just follow the protocol. Nevertheless I would like to add two informations:

There are two controls included in the kit: positive and negative (methylated DNA and unmethylated DNA) and two primers pairs for these controls. You can definitively use these controls with the primers during the protocol.

But there are two others primers included - Alpha X1 (or TSH2B) and GAPDH, which are human specific, so you should replace them by the primers specific for bacterial region of interest.

What is maximum amount of genomic DNA that can be used in a single IP?
Diagenode recommends 1 µg of DNA per IP assay, in our standard protocol from the MeDIP kit. The amount of DNA and antibody per IP as well as the amount of DNA controls to add per IP has been optimized for better specificity. For experiments requiring more than 1 µg, we recommend to perform replicates and then pool the DNA after IP step (as suggested in the manual). If you use more DNA in one IP tube, the amount of the other compounds will have to be titrated, especially the antibody.
How many reactions can be purified with a single IP module (mcgreen-01)?

Ten IPs can be done with one IP module.

Is it possible to freeze the cells immediately after the collection and work with them later, after thawing?

The best is to work with fresh material, because we never know what influence on biological sample can have freezing/thawing. However, if you need to freeze your cells before performing your immunoprecipitation, we recommend that you compare the effects of freezing a sample to the results from a fresh sample. This means that you should perform the MeDIP with fresh and frozen samples in parallel and compare the results to account for any variation in.

We do not recommend using frozen cells without comparing them with freshly collected cells!


Cell lysis and DNA fragmentation

Can the Bioruptor® Sonicator be used to lyse cells instead of using the lysis buffer which is included in the MeDIP kit?
We do not recommend using the Bioruptor® Sonicator for cell lysis. Since the complete lysis buffer for DNA extraction contains proteinase K, the protocol allows for both cell lysis and protein digestion. Following phenol-chloroform extraction and ethanol precipitation allow getting pure genomic DNA and the quality of DNA is important for MeDIP results. Moreover, it is easier to shear pure DNA rather than chromatin. Note that it is possible to use Bioruptor® Sonicator for cell lysis for other purpose (for example for cell lysis for Western Blot analysis) but in the case of MeDIP we recommend to use the proposed protocol starting with genomic DNA extraction and followed by shearing.
Is it possible to fragment genomic DNA with a restriction enzyme protocol instead of a sonicator?
Yes it is possible, as long as the digestion efficiency is assessed and as results you get the DNA fragments about 200-500 bp.
The downstream application I plan to use after IP needs slightly larger fragments than those you suggest as the optimal shearing length; - perhaps up to 1100 bp. Do you know what effect this may have on the efficiency of IP?
Diagenode recommends to shear the DNA to 200-500 bp fragments. It is possible that larger fragment sizes can increase the background. However, we have never tested it in our lab.
What is the optimal range of DNA fragment size after sonication?
The average range after sonication is between 200-500 bp.
What is the amount of sonicated DNA is needed to effectively visualize fragmentation on an agarose gel?
The MeDIP kit manual recommends to use not more than 5 µg of sonicated DNA per line. The migration of large quantities of DNA on agarose gel can lead to poor quality pictures, which do not reflect the real DNA fragmentation. We recommend optimizing and/or standardizing the required amounts based on your specific observations.
What happens after DNA shearing? DNA will be denatured and degraded as well?
DNA shearing leads to DNA fragmentation, but not to degradation. You should get fragments of average size about 500 bp. Sheared DNA has to be denaturated in order to facilitate antibody binding to 5-methylcytidin (steps 4 and 5 in the protocol).

Bead, Antibody and Immunoprecipitation - related questions

Which beads are used in Methyl DNA IP? Can I use alternative beads?
We utilize protein A/G beads which are blocked to avoid the background. These beads are pre-blocked and optimized for highest signal-to-noise ratios for methylated DNA immunoprecipitation. Due to potential issues of high background signal, we do not recommend using other beads unless they are properly pre-blocked and optimized for use in methylated DNA immunoprecipitation.
How long I can store the once blocked agarose beads from the kit?
The agarose beads provided in the MeDIP kit are already blocked and ready to use. Store at 4°C, no dot freeze! Expiration dates are depends on production dates and they are indicated.
Do I need to mix bead tube by hand shaking before using or just pipette the beads without disturbing?
Beads should be mixed gently (this important) by hand shaking or pipetting before using. You should avoid vortexing.
I am working with the MeDIP kit and the vial with beads is getting finished. There is still about 50 µl in it, I guess, but the problem is I cannot get all the beads out because there is only a small amount of fluid left. Could you please tell me in what solution these beads are? If I could redissolve the beads in a higher volume, I could still recuperate them all.
You can prepare 50% suspension using 1x buffer A from the kit (provided buffer A is 5x).
During the wash step, removing the supernatant we are not sure if we take some beads with it or not (since the beads are transparent). Do you recommend some easy trick to avoid this problem?
As the sepharose beads are transparent and may be difficult to see, it is best to use transparent colourless tubes for best separation results.
What is the concentration of antibody in the kit? The kit suggests to use 0.3 µl. If I order separately MAB-5MECYT-100 (1 µg/µl), then how much do I need to add for one IP?
Antibody concentration is the same in the kit and in separate format: 1µg/µl.
What is the ratio between the DNA and antibody?
The ration antibody/DNA in MeDIP is very important parameter in order to get highly specific results. Increasing antibody amounts results in less specific results. The ratio recommended in the kit (0,3 µl for 1 µg of DNA) is optimized.
How long I can store antibody from the MeDIP kit at -20°C? How long can I store MAB-5MECYT-100 from my separate order at -20°C?
5-methyl-cytidine antibody (separate or from the kit) is quite fragile, so the best way is to storage at -80°C. Aliquot the antibody and avoid multiple freeze-throw. It can be stored at -20°C for 1 month. Do not store at 4°C, it degrades rapidly.
How long can I store the aliquoted antibody at -80°C?
The antibody is stable at least for 1 year at -80°C. Aliquot the antibody and avoid multiple freeze-throw.
We would like to use the antibody anty-5-methyl-cytidine to immunoprecipitate genomic DNA from mouse brain tissue for a MeDIP assay. My concerns are about the cross-reactivity of this antibody since it was raised in mouse and we will be using it to probe mouse tissue.

In the data sheet for anty-5mec you can find the information: Specificity: Mouse: positive. We have tried MEDIP with the antibody in mouse followed by QPCR with positive and negative control primers and got the expected results.
(http://www.diagenode.com/media/catalog/file/MAb-006-100-lot-DA0019_09_11.pdf)

You can find the results of our MeDIP experiment in the datasheet of the positive control primers (pp1042).
(http://www.diagenode.com/media/catalog/file/PP-1042-050_lot 001_06_11.pdf)

Also, it is a monoclonal antibody that is directed against methylated cytosine in DNA; we would not expect it to cross-react. So taking all this information together we would recommend this antibody for your application. Unfortunately I do not have any references for this antibody being used in mouse tissue.

In MeDIP short protocol (page 10: STEP 4 and 5: #4), what is the input sample?
An input sample corresponds to DNA that is not used for IP but it is prepared in the same way as IP mix. For more details, see detailed protocol (p.21, step 20): briefly, IP mix contains buffers, meDNA and unDNA internal controls as well as DNA of interest. 20% of excess should be added and kept as Input (15 µl). You need both, IP’d sample and Input for qPCR analysis as the final goal is to calculate the enrichment in IP samples vs Input. Input and IP samples are prepared in the same IP mix. An aliquot of this mix is taken and kept as Input. The goal is to manipulate in the same way IP and Input as much as possible. At purification step, Input and IP are treated in the same way, with the same reagents and volumes.
Adding DNA to IP incubation mix (STEP 4), is it possible to add more than 10 µl of sample? Like for example 40 µl of DNA (concentration 25 ng/µl)?
It is possible to use bigger volume for DNA adjusting water added to IP reaction. Note that the final volume of IP mix should be kept as the ratio of all components is important. Otherwise, buffers concentration changes that can affect the efficiency of immunoprecipitation. If the volume of IP mix changed, it can result also in beads dilution what decreases the efficiency of IP.

Internal Control - related questions

How much methylated control should be used per IP?
Please follow the optimized guidelines provided in the MeDIP kit protocol (Step 4) for the amount of control to add. We do not recommend changing the conditions as the protocol was optimized for better specificity.
How much of DNA - positive and negative control - we add to each IP?
The quantity of DNA which we add as a control is 0,25 ng of each (both positive and negative).
Do the methylated and unmethylated DNA controls interfere with the experimental input DNA that will be immunoprecipitated?
No. It does not interfere with any kind of DNA except the DNA from Arabidopsis, because the Methylated DNA and Unmethylated DNA are sheared DNA from Arabidopsis thaliana. After the shearing, the corresponding DNA has been enzymatically modified with Sss I methylase in vitro in order to produce the Methylated DNA.
Performing qPCR on my IP samples (with or without the control spike DNAs), should I see at least the same ratios using the new rat primers as that of the control DNAs using the control primers? When adding the control DNAs in my samples, can I perform qPCR using the control primers as well as the new rat primers for QC of the IP samples before microarray? Can I expect to see the same ratios between methylated and unmethylated controls (spike DNAs and rat genomic DNAs)?
The presence of spike DNA in IP sample allows to control the efficiency of each IP independently of your DNA of interest. For example, if you have a good ratio methylated DNA:unmethylated DNA with control spikes but not with the DNA of interest that means that your kit still works well and the problem is probably with your DNA (unpure, not sheared etc). Moreover, it is possible to have a situation where methylation status of genomic loci used as positive/negative in rat genome change depending on experiment design (treatments etc); so in this case the only way to check IP is the use of spike DNA.

DNA purification - related questions

What are MeDNA-IP co-precipitant and meDNA-IP precipitant? They are reagents for precipitating IP DNA, not spike DNA?
DNA precipitant and co-precipitant are used for facilitate DNA precipitation at low concentrations. They will precipitate any kind of DNA, IP or spikes.
For recovery of the immunoprecipitated DNA, can I get better results with a phenol chloroform extraction instead of a column extraction?
The efficiency and yield of the DNA extraction is typically higher using a phenol chloroform extraction. However, same people still use column extraction to get the DNA for downstream applications such as PCR or T7 amplification for microarray (ChIP-chip) analysis. Also on this way our kit was validated for microarray analysis. Pooling multiple samples prior to DNA purification may improve yields for column extractions.
Can I use the Zymo kit for DNA purification after the immunoprecipitation?
As the DNA purification step is critical and the loss of precious DNA might occur at this step, we recommend our optimized protocol from the MeDIP kit. Recently we have compared different DNA purification methods of immunoprecipitated DNA. We can conclude that Phenol/chloroform & ETOH precipitation dramatically improved our yield (93%) of methylated product (qPCR data) compared to the use of Zymo (43%) and Qiagen (52%) columns.
According to the MeDIP kit protocol, if I followed DNA purification step for my isolated DNA, 600 µl PB or 1 vol. of phenol/chloroform/IAA should be added into the tube containing agarose beads. Is this right?
In the case of phenol-chloroform extraction, 2 options were tested in our lab: adding phenol directly to the tube with agarose beads and phenol-chloroform extraction from eluate without beads. No difference was found.
After IP, I expect to get 20 - 50 ng of DNA by column elution in a final 30 µl EB buffer (PCR purification kit). How do I check this small amount of DNA concentration in order to know how much do I get?
DNA concentration can be measured by Nanodrop. Keep in mind that recovered DNA is in single stranded conformation - choose corresponding option at Nanodrop. Consequently, fluorophore-based DNA measurement (as Picogreen) is not suitable as it specific for dsDNA.

PCR & PCR Control - related questions

Can the efficiency of the immunoprecipitation (testing the positive and negative controls) be assessed using endpoint PCR instead of qPCR?
Yes, it is possible but quantification ability will be limited. Generally we recommend to use qPCR for data analyse, because this method allows to compare quantitatively your IP samples versus INPUT sample. However if it is not possible to use qPCR, but only end-point PCR, it is better to use semi-quantitative PCR to be able to estimate the quantity of the products.
What are the PCR controls included with the kit? What are the final amplicon lengths for the control PCR fragments?
The following primers sets are provided in our MeDIP kit:
Control name Description Amplicon size
meDNA
positive control 1
The primer pair for this control is specific to a CpG region of the Methylated DNA internal control. The Methylated DNA is sheared DNA from Arabidopsis thaliana. After the shearing, the corresponding DNA has been enzymatically modified with Sss I methylase in vitro in order to produce the Methylated DNA. The use of this control does not interfere with PCR amplification from mammalian cells and allows to control the efficiency of the immunoprecipitation. 1bp
meDNA
positive control 2
The primer pair for this control is specific to a CpG region of the Methylated DNA internal control. The Methylated DNA is sheared DNA from Arabidopsis thaliana. After the shearing, the corresponding DNA has been enzymatically modified with Sss I methylase in vitro in order to produce the Methylated DNA. The use of this control does not interfere with PCR amplification from mammalian cells and allows to control the efficiency of the immunoprecipitation. 87bp
unDNA
negative control 1
The primer pair for this control is specific to a CpG region of the Unmethylated DNA internal control. The Unmethylated DNA is sheared DNA from Arabidopsis thaliana. 84bp
unDNA
negative control 2
The primer pair for this control is specific to a CpG region of the Unmethylated DNA internal control. The Unmethylated DNA is sheared DNA from Arabidopsis thaliana. 92bp
humGAPDH The humGAPDH primers span the human GAPDH promoter sequence that is known to be unmethylated. This control tests immunoprecipitation efficiency. The amplified fragment is located in chr12: 6513913+6513993. 81bp
HumAlphaX1 The humAlpha X1 primers span the human X-linked alpha satellite DNA that is known to be methylated. This control tests immunoprecipitation efficiency. Please see locus X02418 in the NCBI sequence viewer. 102bp
HumTSH2B
(available separately at Diagenode as a positive control)
The region amplified with TSH2B primer pair corresponds to genomic locus which is highly methylated in all somatic cells. This control tests immunoprecipitation efficiency. The amplified fragment is located in chr6:25835060-25835229. 170bp

Diagenode does not provide the sequences of the primers.

Note, that the control primer pairs for mouse and rat are availably separately:

  • as positive control: TSH2B (mouse and rat)
  • as negative control: GAPDH promoter (mouse and rat).
Do the qPCR primers in the MagMeDIP and MeDIP kits work for mouse samples as well as human?
No, the primers included in the MeDIP and MagMeDIP kits are human-specific. Nevertheless the primers for mouse and rat are available at Diagenode. Ordering the kit please mention the organism which you work with and Diagenode will send you the kit with the correct primers.
What pair of qPCR primer sequences can validate the immunoprecipitation efficiency of mouse samples?
Diagenode propose 4 pairs of primers: 1) The primer pairs for both kit internal controls: methylated DNA (meDNA) and unmethylated DNA (unDNA) controls. Provided primers are specific for the kit control; that is why they can be used in mouse also. 2) As negative control: GAPDH promoter (mouse); ref: pp-1045-050; -500 3) As positive control: TSH2B (mouse); ref: pp-1042-050; -500
Can Diagenode provide the primer sequences for the MeDIP kit controls?
Diagenode does not provide the sequence information for the control primers. However, please see the FAQs on controls to obtain information on the amplicon and amplified region coordinates.
For the amplification step, why has the 72°C extension step been omitted?

The extention step at 72°C has been omitted because the amplification with provided primers occurs efficiently during short interval of time, when the Termocycler rises the temperature from 60°C to 95°C. Note, that the extention step at 72°C can be added without affecting the qPCR results. In this case we propose following PCR programme:

  • STEP 1) 95°C - 5min
  • STEP 2) 40 cycles of 95°C - 30s 60°C – 30s 72°C – 30s
  • STEP3) 72°C – 2min
Should I use TaqMan or Sybr Green in the qPCR module of the MeDIP kit?

We have optimized the module with Sybr Green. TaqMan has not been tested in our lab. Nevertheless both can be used as follows:

  • Sybr Green: following our protocol, using our primers (Sybr Green on DNA sample and internal controls)
  • If you prefer TaqMan, you should use your own TaqMan primers with your DNA sample, but not with the internal control.
How do you generally interpret the results from the qPCR validation of the immunoprecipitated DNA? How can I interpret the Ct values?

The interpretation of the results depends on the analyzed locus. Generally we can say that for negative locus Ct value of Input is lower than IP, because there is no enrichment.
But for positive locus Ct values can be at the same range or even Ct value for IP can be before Input, depending on the enrichments.

The Ct values can be used for the calculation of efficiency of MethylDNA Immunoprecipitation and reported as % of immunoprecipitated DNA to Input DNA. The quantification is based on the comparative Ct method and involves the comparison of the Ct values of the IP to the Ct values of the Input:

% (ChIP/Input)= 2^(CtINPUT- CtIP)*100% (1)

Here, 2 is amplification efficiency (E=2). For the calculation to be valid, the amplification efficiency with given primers pair has to be close to 100% meaning that for each cycle the amount of product is doubles (E=2).

Input sample recovered after MeDIP represents only 20% of starting material. Consequently, Ct value of the Input obtained after qPCR corresponds to 20% of Input (5x dilution) but not to 100%. Therefore, an introduction of a compensatory factor in the formula (1) is needed in order to correct Ct of diluted Input.

Compensatory factor corresponds to log2(X) where 2 is the efficiency E=2 and X is the Input dilution.
For 5x dilution, compensatory factor corresponds to log2(5)=2,32

% (ChIP/Input)= 2^((CtINPUT- 2,32)-CtIP))*100%

I observe extremely high Cts for the immunoprecipitated sample (positive locus) and for my internal controls comparing with the Cts for the input DNA. What does it mean?

Analyzing your description we can conclude that the PCR did work but the DNA recovery from the immunoprecipitation was too low. Low enrichment is also confirmed by the high Ct values for the controls.
To avoid the problem of low IP recovery:

  • Use the correct concentration of antibody as recommended in the manual.
  • Double check the amount of DNA and antibody
  • Use the recommended ratio of antibody to DNA – the ratio is critical.
  • Always use fresh dilutions of antibody (within the same day).
  • Avoid excessive freeze-thaw of the antibody – you may aliquot the antibody upon receipt, freeze the aliquots, and prepare fresh dilutions before each use.
  • Assure that the purification of the immunoprecipitated DNA is as recommended in the manual.
  • Always remember to add Buffer B (from Table 5 in the manual) to achieve good immunoprecipitation.
Do you have any protocol for end-point PCR using meDNA positive and negative control primer pairs in order to see the presence or absence (or very faint band)?

We mainly work with qPCR and not with end-point PCR as the aim is to quantify the enrichment. For end-point PCR, I suggest to run less cycles (25-30) at annealing T 60°C.

  • Step 1. 95°C 5 min
  • Step 2. 25-30 cycles of 95°C - 30sec 60°C - 30 sec 72°C -30 sec
  • Step 3. 72°C 2 min

PCR product of 81 bp is expected for positive control and 92 bp for negative control.

I used end-point PCR to analyse the MeDIP results; I got nice enrichment for positive controls and for my samples and non enrichment for negative control. Can I conclude that my MeDIP was very successful? Usually, how much fold difference do I suppose to get using this kit?

It is quite difficult to quantify accurately data from end-point PCR; qPCR is preferable. However, your data looks well, the enrichment is obvious for positive locus but not for negative. The enrichment that we get is calculated from qPCR and not from end-PCR.

In my MeDIP assay I use H19 as positive control and CTRL as negative control. Using the end-point PCR I see a really nice enrichment for H19, but I also have a slight band after CTRL PCR (33 cycles). When I decrease the cycles to 30, this CTRL signal disappears, and the H19 signal stays, that is good. I also did a no-antibody control and this was as expected negative. 1) Is this slight signal after CTRL PCR due to unspecific binding of the 5-methyl-cytosine antibody? 2) Should I do an IgG control?

When using end-point PCR it very often happened that you amplify something, you get the band where you do not expect it, depending on the number of cycles. It is quite difficult to quantify the results obtained with this method. qPCR allows to compare quantitatively your IP samples versus INPUT sample. That is why we recommend to use qPCR for data analyse. Some information about data analyse after qPCR you can find on our website, par example in the manual of MeDIP kit. If you can not run qPCR, please do semi-quantitative PCR.

Please, look at the comments:

Answer to the Q1) Not necessary. It is possible that even the negative control like CTRL gene might contain some methylated CpG. Moreover, even if the region amplified with specific primers was not methylated itself, it is possible that adjacent regions include some meCpG. It will results in some enrichment as immunoprecipitated DNA is randomly sheared before MeDIP.

Answer to the Q2) No, it is not necessary. We never perform IgG control working with MeDIP.


DNA quality, characteristics and enrichment after immunoprecipitation

After immunoprecipitation, is the DNA single-stranded or double-stranded?
DNA is single-stranded.
What percent enrichment of methylated DNA should be expected after immunoprecipitation?

The fold enrichment will depend on your target DNA. See below some results obtained with different DNA samples, with different DNA target regions.



In addition, see the enrichment values obtained for the different MeDIP Kit controls below:

What is the typical yield of the immunopreciptated DNA?
The recovery after MeDIP can vary for different cell type but an average recovery is between 20-50 ng per IP.
Note that recovered DNA is in single stranded conformation. Measuring the DNA concentration choose corresponding option at Nanodrop. Consequently, fluorophore-based DNA measurement (as Picogreen) is not suitable as it specific for dsDNA.
What steps can I take to get best possible enrichment from methylated DNA immunoprecipitation?
  • Use the correct concentration of antibody as recommended in the manual.
  • Double check the amount of DNA and antibody
  • Use the recommended ratio of antibody to DNA – the ratio is critical.
  • Always use fresh dilutions of antibody (within the same day).
  • Avoid excessive freeze-thaw of the antibody – you may aliquot the antibody upon receipt, freeze the aliquots, and prepare fresh dilutions before each use.
  • Assure that the purification of the immunoprecipitated DNA is as recommended in the manual.

Always remember to add Buffer B (from Table 5 in the manual) to achieve good immunoprecipitation.

After MeDIP and column purification, I got less than 20 ng of DNA. What is an expected yield after column purification, 14 ng - 33 ng per IP?
The recovery after MeDIP can vary for different cell type but an average recovery is between 20-50 ng per IP when DNA is purified by phenol-chloroform extraction. Columns-based purification results in some DNA lost comparing to phenol-chloroform extraction. The values you obtained seem to be correct.
Is it possible to use the same concentration of the isolated DNA as the input DNA for qPCR or DNA amplification followed by DNA labelling? Does it make a big difference?
The data analysis after MeDIP consists of the calculation of the enrichment of immunoprecititated DNA vs Input DNA. Data are reported as % of immunoprecipitated DNA to Input DNA. Therefore, it is important to keep existing ratio between IP and Input. Any changes in DNA amounts will lead to “false” results. Moreover, DNA amounts obtained after MeDIP is quite low making them difficult to accurate quantification.

Alternative uses / differences from other protocols for the MeDIP kit

I am using the antibody anty-5methyl Cytidine for methyl DNA IP (in house protocol) and I was wondering what is your recommendation for the ratio of antibody to DNA? So far, I have tried 5 µg antibody: 4 µg DNA and it seems to work fine, but I'm wondering if I should use less antibody.
We recommend the use of our kit and protocol as the DNA:antibody ratio has been carefully optimized. It is critical not to use an excessive amount of antibody. The lack of antibody can result in low IP efficiency whereas large excess of antibody might lead to lower specificity. You could try 1 ug of DNA per reaction, pool then 4 tubes after IP, if you need to use 4 ug DNA. Titrate the antibody down: you have been using 1.25 ug of antibody per ug of DNA; less is used in the kit, because the ratio has been optimized in house.
Is it possible to use the MeDIP kit to study the methylation of guanine residues at O6?
Due to antibody which is used in the kit, MeDIP kit is specific for 5-methyl cytidine. This antibody does not recognise other modification.
How does the immunoprecipitation protocol used with the MeDIP kit differ from the protocol used in the paper by Weber et. al. (2005). Nat. Genet. (38) 7: 853-862?

The Diagenode protocol in the MeDIP kit is different from the protocol used in the Weber et. al. paper in several aspects. The MeDIP kit contains an optimized buffer system for higher antibody specificity.
Specifically, we outline the differences below:

  • The MeDIP kit uses a number of different buffers for the immunoprecipitation and bead wash steps instead of the single buffer used in Weber’s work.
  • The beads in the MeDIP kit are not magnetic as in Weber´s paper.
  • The elution step is different from described by Weber.
  • The recommended antibody incubation time in the MeDIP kit is longer for optimal results.
  • The amounts and the ratio of genomic DNA and antibody differ between the two protocols, as the MeDIP kit has been optimized for highest sensitivity and specificity with the antibody.
I would like to study the methylation status of 3 miRNA of known sequences. Important: this study has to be done on DNA extracted from samples paraffin included. Can Diagenode´s MedIP or MethylEasy kit be used for this approach?

It is not clear in your question whether you want to study DNA Methylation of RNA or DNA?
To answer to your question:

  1. The 5-methyl cytidine antibody of the MeDIP Kit detects methylated cytosines, so it should also recognize 5-mec in RNA.
  2. Bisulphite method can be used only with DNA. RNA is degraded rapidly by bisulphite.
  3. Paraffin-embedded samples can be used for MeDIP, but:
    1. DNA is fixed very strongly, so it is difficult to purify it. Unpure DNA can introduce bias. Note also, that the fixation introduces DNA modifications (addition of mono-methyl group, formation of bridges between adjacent bases, generation of apurinic and apyrimidinic sites and strand breaks), what can cause the problem for efficient PCR.
    2. DNA is already fragmented, so it does not have to be sonicate. But the size of the fragment should be checked, par example on agarose gel. This method can cause the problem since a little material is disponible when working with paraffin-embedded samples. Alternatively it is possible to use the BioAnalyzer from Agilent (little material required). We have never tested paraffin-embedded samples with the MeDIP kit in our lab.
  4. Paraffin-embedded samples can be used for Bisulphite, but: a) samples are already fragmented and normally the unfragmented DNA is used in bisulphite method as the bisulphite treatment degrades DNA. So you are not in optimal conditions.
What is a difference between MeDIP kit vs MagMeDIP kit (DNA purification vs no DNA purification)?
The protocol proposed in MagMeDIP kit is faster as due to magnetic beads wash steps are shorter. The composition of DNA isolation buffer is different from elution buffer in MeDIP kit. It allows the direct use of isolated DNA in qPCR without additional purification. Anyway, for other sensitive applications like amplification, DNA should be purified. You need to perform the complete protocol: genDNA isolation, fragmentation, immunoprecipitation, elution and purification. It is recommended to analyse MeDIP by qPCR before starting microarray study.

Amplification

When should I consider amplifying my immunoprecipitated DNA?
For the optimal performance of high throughput genomic technologies sufficient yields of high-quality DNA are crucial. The amount of DNA isolated per MeDIP is about 20 to 50 ng. It is possible to pool several samples to get enough DNA and avoid amplification. However, if multiple experimental points are to be analyzed or if primary cells or tissue samples are to be used, the number of cells can be a limitation and amplification of immunoprecipitated material is necessary.
What amplification methods can be used after the MeDIP?

There are many compatible methods for amplifying DNA from low starting amounts of DNA (as in ChIP and Methyl DNA IP) including TLAD, WGA, LM-PCR, etc.

Amplification of DNA is a method that takes template DNA and makes many copies of it; PCR is one prime example. However, methods such as PCR are exponential amplification methods, meaning that the number of copies made of the template DNA increases at an exponential rate. The largest shortcoming of exponential amplification approaches is their diminished fidelity when amplifying complex mixtures of DNA. In an exponential amplification, variances or fluctuations in the amplification efficiency of a given amplicon early in the amplification will themselves amplify to considerable and detectable differences at the end of the amplification, because of the exponential kinetics. In linear amplification, however, the linear amplification kinetics minimizes such variances in outcome relative to exponential amplification.

In addition:

  • Whole genome amplification (WGA): It was shown that the signal to-noise ratio obtained in ChIP-on-chip using WGA method of amplification of immunoprecipitated material is superior to LM-PCR.
  • T7-based Linear Amplification of DNA (TLAD): TLAD begins with a terminal transferase (TdT) tailing of the template DNA, is followed by annealing with a primer adapter containing the T7 promoter sequence. A second strand is synthesised with DNA Polymerase I Klenow fragment, and then in vitro transcription of the template generates a RNA amplification product (Liu et al, 2003). This results in linear rather than exponential amplification, and this method has the potential to reduce potential bias introduced by PCR-based amplification. However, it is very expensive, tedious, and time consuming.

In our lab we have tested T7 protocol which works really well with the samples after MeDIP. That is why we can recommend this method for amplification.of MeDIP samples. Also from the feedback from our clients we know that WGA method is compatible with DNA samples obtained after MeDIP.

Diagenode recommends TLAD as a method for DNA amplification after IP. However we have previously used a Sigma WGA kit with our experiments before hybridizing on an array; is this method compatible with your MeDIP kit?
It is compatible, please see answer to the question above.
Spike control will be amplified by WGA2 kit (Sigma)?
Spike control as well as DNA of interest will be amplified. Spike control it is no mammalian DNA and it does not interfere with microarray.
What amount of DNA do you need to start with for DNA amplification?
It is depend on the amplification method you use. For T7 you can start even with 10-15 ng per reaction. For other methods – please check the producer information.

Downstream applications: microarrays, sequencing - related questions

What is the amount of DNA recommended for a microarray experiment?

1-2 µg per array has been shown to work well. To get this amount of DNA it is possible:

  • to pool several samples obtained after MeDIP
  • to amplify immunoprecipitated DNA.

After one successful round of TLDA amplification, you can provide up to 4 µg of DNA.

Has Diagenode tested the MeDIP kit for use with microarrays (for DNA methylation analysis) after immunoprecipitating the methylated DNA? What is the recommended amount of DNA and antibody for this downstream application?

Diagenode has tested the MeDIP kit (using our antibody anty-5methyl cytidine) for microarray application. For this downstream application:

  • we recommend the same DNA and antibody amounts as stated in the manual.
  • pool the immunoprecipitated DNA samples or amplify DNA to get enough material for microarray
  • important: do not use more DNA per IP reaction, but more reactions and pool when the IPs are done
Is it possible to use the internal controls from the MeDIP kit for microarray analysis with any commercial microarrays?
Currently, no microarray manufacturer has included the Diagenode internal controls.
What amount of antibody anty-5-methyl cytidine should be used on Nimblegen platform for MeDIP studies?
The quantity of antibody used for MeDIP is important parameter as using much will increase the background. In our MeDIP kit protocol we recommend to use 0,3 µg per IP (for 1 µg of DNA). It yields in 20-50 ng of recovered DNA. Usually, 1-2 µg of DNA are needed for microarray meaning that immunoprecipitated DNA should be amplified (or if enough of starting material, samples can be pooled).
I am planning to do MeDIP and then send the MeDIPed to NimbleGen company for microarray experiments for further processing (http://www.nimblegen.com/products/methylation/tutorial.html).
  1. How to modify the original MeDIP protocol kit from Diagenode for NimbleGen's suggestions (http://www.nimblegen.com/products/methylation/tutorial.html)?
  2. Do you have MeDIP kit for rat? or monoclonal rat antibody for MeDIP?
  3. Can I skip the DNA denaturation step?
  4. After amplification, I would like to send these IP'd samples to NimbleGen for microarray labeling and hybridization. What would be a best way for QC before sending the samples?
  1. You do not need to modify the original protocol of MeDIP from Diagenode for microarray hybridization. You should perform MeDIP as describe in Diagenode manual and proceed the amplification (step 8 in NibleGen protocol). Note that an average recovery after MeDIP with our protocol is about 20-50 ng depending on cell type (from 1 µg of starting material).
  2. Our MedIP kit can be used for human/rat/mouse DNA as the antibody used in the kit recognizes 5’-methylcydidine in DNA from different species. The positive (TSH2B) and negative (GAPDH promoter) control primers for rat are already available at Diagenode.
  3. DNA denaturation step is important for following antibody binding so I do not recommend to skip this step. At the end of MeDIP, DNA can be used for amplification with Sigma WGA2 kit or T7 protocol and hybridization.
  4. The best way is to control IP before and after amplification by qPCR: Control spike DNA (methylated and unmethylated) with corresponding primer pairs are provided in the kit. It allows to check the efficiency of each IP and do not interfere with following hybridization. The second type of control is qPCR with primers for methylated and non-methyl region in genome of interest (rat in your case).
If studying DNA methylation status in rat using MeDIP and then microrray, is it possible to exclude the positive/negative DNA control steps? Some reviewers may challenge these controls because plant genomic DNA region may have a homologous sequence to rat. Is it possible that these plant DNA sequences are a very short probe size that does not have any homologous sequences to any mammalian species?
Our control DNA does not interfere for any mammalian DNA and IP samples can be used for microarray study. Anyway, you can perform immunoprecipitation without control DNA as it used only to check the efficiency of IP. The alternative is to put DNA controls only in one tube which is dedicated to qPCR analysis and to not add the control to the samples going to amplification/microarray assay.
For methylation microarray purpose, which protocol for DNA elution and purification is recommended? Is this a short protocol (page 10 and 11) or detailed protocol option 2, not option 1?

DNA samples immunoprecipitated using MeDIP or MagMeDIP kit can be used successfully for microarray analysis. An average recovery after MeDIP (starting material is 1 µg) is about 20-50 ng of single-stranded DNA. As microarray study requires 1-2 µg of DNA, immunoprecipitated DNA should be amplified.

In the case of MeDIP kit (cat # mc-green-003), isolated DNA can be used directly for any down-stream application; you do not need to perform additional purifications. According to this protocol immunoprecipitated DNA is first eluted from the beads and then purified. Two options for DNA purification are proposed: phenol-chloroform extraction or columns-based purification. Both methods result in pure DNA of sufficient quality. Columns-based method is faster but about 30% of DNA is lost during this step.

If you work with MagMeDIP (cat # mc-magme A10 or A32), DNA isolated with DIB buffer can be used directly for qPCR but for other applications it is better to not use DIB buffer, but to perform additional purification: elution with our DNA purification module (cat # mc-magme-002) with following phenol-chloroform extraction or columns purification. Note anyway that columns purification results in significant DNA lost (about 30%).

After the DNA purification, using either the quick (buffer DIB) or the slow (complete elution buffer D, E and F from the DNA purifying module) protocols the amount of DNA that I get at the end is too low or too dirty to use in further Illumina sequencing. So I have few questions. - How do you check that you have DNA after your IP? I did a PCR on the positive control MeDNA with the specific primers furnished in the kit and I got some amplification (proof that there is some methylated DNA). But, if I quantify that DNA its concentration is less than 2ng/uL which is way to low and the 260/280 ratio is about 0.8 which means that the DNA obtained is too dirty to send into the Illumina sequencing (which is what I would like to do).

The Illumina sequencing required double stranded DNA; after MeDIP the DNA is in single stranded confirmation, so the MeDIP protocol has to be modified. Diagenode proposes the protocol for MeDIP-seq (please, look at the question/answer below) according to which the adaptors have to be ligated to DNA BEFORE MeDIP what will allow to use Illumina protocol.

Concerning DNA purification, you mentioned that you were using two methods: DIB purification and DNA purifying module with phenol-chloroform extraction. DIB purification results in DNA purity sufficient for PCR analysis but for any other applications; DNA should be additionally purified. For the moment there are 2 option for DNA purification - by phenol-chloroform extraction (what you did) or column-based (note that the second results in DNA lost). Note also that Diagenode has been working on the development of new magnetic DNA purification system and it will be available soon.

Concerning your results: DNA recovery after MeDIP varies from 20 to 50 ng per IP depending on cell types. You wrote that the concentration of your sample was around 2 ng/µl, this means that in your total sample (50 µl) you got around 100 ng. So this is correct with what we could expect. DNA quantification after MeDIP is quite difficult as the amount is at the limit of detection for Nanodrop. It is possible to dissolve DNA pellet after phenol-chloroform extraction in a smaller volume to get more concentrated solution. If you wish to have more purified DNA, you can perform replicates and then pool them after IP. Note also that recovered DNA is in single-stranded conformation. The fluorofore-based quantification is not possible as this method requires dsDNA. The possible option for MeDIP-seq is the amplification of DNA recovered after MeDIP, using one of the methods disponible (for example T7-based Linear Amplification of DNA).

What MeDIP-Seq protocol recommendations do you recommend?

The main steps for MeDIP-seq are quite simple but there are some variations. Diagenode has discussed the MeDIP-seq protocol with our expert customers. The following describes conditions tested by our customers for highthroughput sequencing using an Illumina system.

(1) Diagenode recommends sonicating the DNA so that the peak of material is quite short (150-250 bp); using the Diagenode Bioruptor® Sonicator, gDNA can be sonicated using the following protocol:

Total time: around 40-50 min,
Cycles: 30s ON, 30s OFF,
Power settings: LOW
Tubes: 0.65 ml silinonized tubes, Costar 3206
Volume in the tubes: 100 µl
Cooling system is recommended in order to get less sample-to sample variation.

(2) Diagenode recommends following the Illumina protocol for library preparation. Specifically, complete the end repair, A-tailing, and adaptor ligation. Some adaptations are detailed in Quail et al (2008) Nature Methods, 5 (12), pp1005-1010, which may be worth considering.

(3) Following library preparation (and clean-up) quantify the library using a DNA measurement device such as a Nanodrop or equivalent. Because the library's concentration is elevated due to the presence of 2x30 bp adaptors, our customer’s recommend adjusting the concentration as follows (Diagenode recommends you independently determine whether this step is necessary or not):

e.g.: (average size of sample [bp] + sum of adapter size [bp]( / (average size of sample [bp]( = elevation constant (175 + 60) / (175) = 1.343
100 ng/µl / 1.343 = 74.47 ng/µl
Subtract 10% from the above to account for self-ligated adaptors
e.g.: 74.47 ng/µl - (74.47ng/µl * 0.12) = 65.53 ng/µl

(4) Use this concentration to add 1.2 µg to the MeDIP incubation mix, which is then performed using the IP-Star and Diagenode's Auto MeDIP protocols.

(5) Perform QC using qPCR for methylated and unmethylated controls that were added during the preparation of the incubation mix. Following the qPCR, purify the MeDIP’d DNA using the Diagenode’s DNA purification Module (mc-magme-002) and Phenol / Chloroform / Isoamyl alcohol or IPure DNA purification protocol.

(6) Following the general DNA purification step, the correct size of DNA of the input DNA and ‘MeDIP’d’ DNA is selected by excising DNA bands (50-350 bp) from a low melting point agarose gel. After clean-up of the size-fractionated DNA from the agarose gel, perform the adaptor-modified PCR. An alternate approach is to perform the adaptor-modified PCR, followed by the size exclusion on the agarose gel. The potential advantage is the higher concentration of DNA obtained after PCR. However a bias may be introduced as different insert sizes amplify at different efficiencies and this should be verified before modifying the recommended sequencing protocols.

(7) Following clean-up the MeDIP is ready to be sequenced

I would like to use DNA samples obtained with Diagenode´s MeDIP kit with the Illumina sequencer, but my samples need to be dsDNA. I could use the T7-Based Linear Amplification of DNA protocol but we will like to avoid any amplification as one of the advantages of the Illumina seq technique is to use very small quantities of DNA. How to obtain dsDNA without the use of any adaptator?

Concerning your question, there are 3 possibilities:

  1. As you know after MeDIP immunoprecipitated DNA is in single-stranded confirmation. If you use the T7-Based Linear Amplification of DNA you will get dsDNA.
  2. it is possible also to ligate the adaptor to DNA BEFORE MeDIP what will allow to use Illumina protocol.(the protocole for MeDIP-seq, please look at the question above)
  3. To get methylated fragments of DNA is it possible to use another method - MBD (instead of MeDIP).
    Using this technique you end-up with dsDNA:
    http://www.diagenode.com/media/documents/H6-GST-MBD_Sheet_1208V2.pdf


Sample types and amount of DNA

Is the MagMeDIP kit considered usable for the FFPE tissue (Formalin-Fixed Paraffin-Embeded)? Has anyone ever published using your kit and FFPE tissues? Would we simply skip the shearing process in your protocol since our DNA will already be smaller than 300 bps?

As you mentioned, DNA from Formalin-Fixed Paraffin-Embeded is already fragmented. If the fragments are of optimal size (300-500 bp), no additional fragmentation is required. It is possible to perform MeDIP but note, that the quality of FFPE DNA is not optimal for MeDIP. The fixation introduces DNA modifications (addition of mono-methyl group, formation of bridges between adjacent bases, generation of apurinic and apyrimidinic sites and strand breaks). DNA quality varies from sample-to sample.

We have never tested FFPE tissue with the MagMeDIP kit in the lab.

Is the MagMeDIP kit suitable for comparing DNA methylation of bacterial DNA?

Our MagMeDIP kit can be used for the DNA methylation studies in different organisms. In the kit we use the antibody raised against 5-methyl-cytosine. In bacteria the methylation happens on cytosine and adenosine residue; so our kit can be used only for cytosine methylation studies.

If you decide to use this kit (so if the methylation concern the cytosine) you can just follow the protocol. Nevertheless I would like to add two informations:

There are two controls included in the kit: positive and negative (methylated DNA and unmethylated DNA) and two primers pairs for these controls. You can definitively use these controls with the primers during the protocol. But there are two others primers included - TSH2B and GAPDH, which are human specific, so you should replace them by the primers specific for bacterial region of interest.

Your manual suggests to being with 200 mg – 1 g of tissue. For human tumor samples this is monumental; what is the least possible starting amount required?
You need 1 µg of DNA per IP as starting amount of DNA
What is maximum amount of genomic DNA that can be used in a single IP?
Diagenode recommends 1 µg of DNA per IP assay, in our standard protocol from the MeDIP kit. The amount of DNA and antibody per IP as well as the amount of DNA controls to add per IP has been optimized for better specificity. For experiments requiring more than 1 µg, we recommend to perform replicates and then pool the DNA after IP step (as suggested in the manual). If you use more DNA in one IP tube, the amount of the other compounds will have to be titrated, especially the antibody.

gDNA preparation for immunoprecipitation

I am starting with gDNA which has been purified using Qiagen’s Dneasy Blood & Tissue kit ( approx size of DNA is 20-50 kbp) and it does use RNAse A to get rid of the RNA and Qiagen says the purity should be okay. Do you have any suggestions about the size and purity of DNA I am starting with?

Any method of DNA extraction resulting in pure genomic DNA (pure DNA has the ratio1.8- 2.0 at A260/280) can be used gDNA preparation for MeDIP. The purity and the size of DNA which you get with Qiagen kit should be OK.

Note that DNA should be quantified before MeDIP to check the purity.

I would like to prepare gDNA using Qiagen kit for isolating genomic DNA. This kit elutes the DNA in Buffer AE( 10mM tris, 0.5% EDTA, pH=9), which I shall shear using sonication to obtain 300-500bp fragments and use for subsequent IP. Will this buffer be okay to use?
We recommend to perform DNA sonication in standard TE buffer pH 8. However you can use this buffer, it should be ok.
What is the optimal range of DNA fragment size after sonication?
The average range after sonication is between 200-500 bp.
Can you recommend the sonication conditions to obtain 300-500 bp fragments?

It is advised to optimize the shearing conditions “in house” with your own sample. Generally we recommend to use 300 µl of your DNA sample resuspended at 0.1 µg/µl (30 µg DNA) in 1,5 ml tube with the settings as below:

  • Bioruptor power: Low
  • cycles: 15 s ON and 15s OFF
  • total time: 10-15min

Usually 10-15min of sonication is enough, but to be sure you need to try and check it yourself.

What is the amount of sonicated DNA is needed to effectively visualize fragmentation on an agarose gel?
The MagMeDIP kit manual recommends to use not more than 5µg of sonicated DNA per line. The migration of large quantities of DNA on agarose gel can lead to poor quality pictures, which do not reflect the real DNA fragmentation. We recommend optimizing and/or standardizing the required amounts based on your specific observations.

Immunoprecipitation - related questions

The magBuffer A form the MagMeDIP kit has precipitated out. I have kept it at the suggested temp (+4°C) and the kit is new. Do you know what is wrong and how I could solve this problem?
This is normal because the magBuffer A contains the SDS, which can precipitate in the low temperature. Please, warm the buffer at RT before using it - it will dissolve the crystals.
In the IP continuation step in your protocol: (pt33) after preparing the diluted AB mix, the step which follows says: Mix and add 20 µl washed beads to each 200 µl IP tubes. Do you add beads using a pipette or just directly? Any suggestions? Again at Pt 34, Place on rotating wheel at 4°C for 4 hrs or overnight. Is there any desired speed for the rotating wheel?

Yes, you should add the beads using the pipette. Resuspend well the beads before each use. Working with the beads please note this: “Keep the beads homogenously in suspension at all times when pipetting. Variation in the amount of beads will lead to lower reproducibility.”

Concerning the rotation wheel, the rotation should be mild, not shaking.

For the Washes (Step 5, Pt 38) you mention to Wash beads again with 100 µl ice cold Mag Wash buffer-2 this. So this has to be done 3 times too, yes? I just wish to confirm that.
Washes, point 37 and 38. No, it is differently. You should perform 3 washes with the Wash buffer 1 (point 37) and 1 washing with the Wash buffer 2. In the manual it is written: “Wash the beads again (as described above: Point 37) with 100 µl ice-cold MagWash buffer-2.” “As described above” – so you should follow the way of washing described in the point 37, but not the number of washes.

DNA elution and purification

For the DNA isolation (Step42) It says to remove tubes from magnetic rack add DIB and resuspend beads and transfer into 1.5 ml tubes. Again for transferring along with the beads is there any suggestion? (I am not sure if you can transfer beads using a pipette)
Add the beads using the pipette. Resuspend well the beads before each use. Working with the beads please note this: “Keep the beads homogenously in suspension at all times when pipetting. Variation in the amount of beads will lead to lower reproducibility.”
What is a difference between MeDIP kit vs MagMeDIP kit (DNA purification vs no DNA purification)?
The protocol proposed in MagMeDIP kit is faster as due to magnetic beads wash steps are shorter. Secondly, the protocol is more flexible concerning DNA purification after IP. The composition of DNA isolation buffer is different from elution buffer in MeDIP kit. It allows the direct use of isolated DNA in qPCR without additional purification. Anyway, for other sensitive applications like amplification, DNA should be purified.
You need to perform the complete protocol: genDNA isolation, fragmentation, immunoprecipitation, elution and purification. It is recommended to analyse MeDIP by qPCR before starting microarray study.
For methylation microarray purpose, which protocol for DNA elution and purification is recommended?
DNA samples immunoprecipitated using MeDIP or MagMeDIP kit can be used successfully for microarray analysis. An average recovery after MeDIP (starting material is 1 µg) is about 20-50 ng of single-stranded DNA. As microarray study requires 1-2 µg of DNA, immunoprecipitated DNA should be amplified.
If you work with MagMeDIP (cat # mc-magme A10 or A32), DNA isolated with DIB buffer can be used directly for qPCR but for other applications it is better to not use DIB buffer, but to perform additional purification: elution with our DNA purification module (cat # mc-magme-002) with following phenol-chloroform extraction or columns purification. Note anyway that columns purification results in significant DNA lost (about 30%).

Control - related questions

What are the PCR controls included with the kit? What are the final amplicon lengths for the control PCR fragments?

The following primers sets are provided in our MagMeDIP kit:

Control name Description Amplicon size
meDNA
positive control
The primer pair for this control is specific to a CpG region of the Methylated DNA internal control. The Methylated DNA is sheared DNA from Arabidopsis thaliana. After the shearing, the corresponding DNA has been enzymatically modified with Sss I methylase in vitro in order to produce the Methylated DNA. The use of this control does not interfere with PCR amplification from mammalian cells and allows to control the efficiency of the immunoprecipitation. 81bp
unDNA
negative control
The primer pair for this control is specific to a CpG region of the Unmethylated DNA internal control. The Unmethylated DNA is sheared DNA from Arabidopsis thaliana. 92bp
humGAPDH The humGAPDH primers span the human GAPDH promoter sequence that is known to be unmethylated. This control tests immunoprecipitation efficiency. The amplified fragment is located in chr12: 6513913+6513993. 81bp
HumTSH2B The region amplified with TSH2B primer pair corresponds to genomic locus which is highly methylated in all somatic cells. This control tests immunoprecipitation efficiency. The amplified fragment is located in chr6:25835060-25835229. 170bp

Diagenode does not provide the sequences of the primers.

Note, that the control primer pairs for mouse and rat are availably separately:

  • as positive control: TSH2B (mouse and rat)
  • as negative control: GAPDH promoter (mouse and rat).
What size fragments would be expected from the amplification of your positive and negative controls?
Amplified region with positive control primer is 81 bp; negative control amplifies a PCR product of 92 bp.
What is the size of the PCR product I should expect using your primers ?( meDNA, unDNA, TSH2B and GAPDH).
The product sizes of the controls are meDNA (81 bp), unDNA (92 bp) and for Human DNA: GAPDH (81 bp), TSH2B (170bp).
How can I calculate a hard value of meDNA control after IP?
The Methylated DNA and Unmethylated DNA are sheared DNA from Arabidopsis thaliana. After the shearing, the corresponding DNA has been enzymatically modified with Sss I methylase /in vitro/ in order to produce the Methylated DNA. The INPUT concentration are 0.2 ng/µl for both and we use 1.25 µl per MeDIP. So after IP, it is difficult to calculate the concentration with a Nanodrop. You can use a standard curve during your qPCR.
Do the methylated and unmethylated DNA controls interfere with the experimental input DNA that will be immunoprecipitated?
No. It does not interfere with any kind of DNA except the DNA from Arabidopsis, because the Methylated DNA and Unmethylated DNA are sheared DNA from Arabidopsis thaliana. After the shearing, the corresponding DNA has been enzymatically modified with Sss I methylase in vitro in order to produce the Methylated DNA.

PCR - related questions

Should I use end-point PCR or qPCR?
It is better to run qPCR rather then end-point PCR in order to get quantitative information.
For the amplification step, why has the 72°C extension step been omitted?

The extention step at 72°C has been omitted because the amplification with provided primers occurs efficiently during short interval of time, when the Termocycler rises the temperature from 60°C to 95°C.
Note, that the extention step at 72°C can be added without affecting the qPCR results. In this case we propose following PCR programme:

  • STEP 1) 95°C - 5min
  • STEP 2) 40 cycles of 95°C - 30s 60°C – 30s 72°C – 30s
  • STEP 3) 72°C – 2min
How do you generally interpret the results from the qPCR validation of the immunoprecipitated DNA? How can I interpret the Ct values?

The interpretation of the results depends on the analyzed locus.
Generally we can say that for negative locus Ct value of Input is lower than IP, because there is no enrichment. But for positive locus Ct values can be at the same range or even Ct value for IP can be before Input, depending on the enrichments.

The Ct values can be used for the calculation of efficiency of MethylDNA Immunoprecipitation and reported as % of immunoprecipitated DNA to Input DNA. The quantification is based on the comparative Ct method and involves the comparison of the Ct values of the IP to the Ct values of the Input:

% (ChIP/Input)= 2^(CtINPUT- CtIP)*100% (1)

Here, 2 is amplification efficiency (E=2). For the calculation to be valid, the amplification efficiency with given primers pair has to be close to 100% meaning that for each cycle the amount of product is doubles (E=2).

Input sample recovered after MeDIP represents only 20% of starting material. Consequently, Ct value of the Input obtained after qPCR corresponds to 20% of Input (5x dilution) but not to 100%. Therefore, an introduction of a compensatory factor in the formula (1) is needed in order to correct Ct of diluted Input.

Compensatory factor corresponds to log2(X) where 2 is the efficiency E=2 and X is the Input dilution.

For 5x dilution, compensatory factor corresponds to log2(5)=2,32

% (ChIP/Input)= 2^((CtINPUT- 2,32)-CtIP))*100%

I observe extremely high Cts for the immunoprecipitated sample (positive locus) and for my internal controls comparing with the Cts for the input DNA. What does it mean?

Analyzing your description we can conclude that the PCR did work but the DNA recovery from the immunoprecipitation was too low. Low enrichment is also confirmed by the high Ct values for the controls. To avoid the problem of low IP recovery:

  • Use the correct concentration of antibody as recommended in the manual.
  • Double check the amount of DNA and antibody
  • Use the recommended ratio of antibody to DNA – the ratio is critical.
  • Always use fresh dilutions of antibody (within the same day).
  • Avoid excessive freeze-thaw of the antibody – you may aliquot the antibody upon receipt, freeze the aliquots, and prepare fresh dilutions before each use.
  • Assure that the purification of the immunoprecipitated DNA is as recommended in the manual.
In the manual of MagMeDIP kit, there are some other hypermethylated gene in MCF7 cells like Cystatin M and Claudin-6 or p14 INK4A intronic CpG Island. Is it possible to get these primers or their sequences?

The primers Claudin-6 and Cystatin M are currently used in the lab but they are not in sale. Note that the high methylation level found in corresponding regions is specific for MCF7 cell line.

The genomic location is chr11:65535926+65536070 for Cystatin M and chr16:3007977+3008125 for Claudin-6. Data on p14 INK4A intronic CpG region were obtained by collaboration and we do not posses the information on primers sequences. Note also that new control primers TSH2B for methylated region are available at Diagenode.

Should I use TaqMan or Sybr Green in the qPCR module of the MeDIP kit?

We have optimized the module with Sybr Green. TaqMan has not been tested in our lab. Nevertheless both can be used as follows:

  • Sybr Green: following our protocol, using our primers (Sybr Green on DNA sample and internal controls)
  • If you prefer TaqMan, you should use your own TaqMan primers with your DNA sample, but not with the internal control.

Amplification

When should I consider amplifying my immunoprecipitated DNA?
For the optimal performance of high throughput genomic technologies sufficient yields of high-quality DNA are crucial. The amount of DNA isolated per MagMeDIP is about 20 to 50 ng. It is possible to pool several samples to get enough DNA and avoid amplification. However, if multiple experimental points are to be analyzed or if primary cells or tissue samples are to be used, the number of cells can be a limitation and amplification of immunoprecipitated material is necessary.
What amplification methods can be used after the MeDIP?

There are many compatible methods for amplifying DNA from low starting amounts of DNA (as in ChIP and Methyl DNA IP) including TLAD, WGA, LM-PCR, etc.

Amplification of DNA is a method that takes template DNA and makes many copies of it; PCR is one prime example. However, methods such as PCR are exponential amplification methods, meaning that the number of copies made of the template DNA increases at an exponential rate. The largest shortcoming of exponential amplification approaches is their diminished fidelity when amplifying complex mixtures of DNA. In an exponential amplification, variances or fluctuations in the amplification efficiency of a given amplicon early in the amplification will themselves amplify to considerable and detectable differences at the end of the amplification, because of the exponential kinetics. In linear amplification, however, the linear amplification kinetics minimizes such variances in outcome relative to exponential amplification.

In addition:

  • Whole genome amplification (WGA): It was shown that the signal to-noise ratio obtained in ChIP-on-chip using WGA method of amplification of immunoprecipitated material is superior to LM-PCR.
  • T7-based Linear Amplification of DNA (TLAD): TLAD begins with a terminal transferase (TdT) tailing of the template DNA, is followed by annealing with a primer adapter containing the T7 promoter sequence. A second strand is synthesised with DNA Polymerase I Klenow fragment, and then in vitro transcription of the template generates a RNA amplification product (Liu et al, 2003). This results in linear rather than exponential amplification, and this method has the potential to reduce potential bias introduced by PCR-based amplification. However, it is very expensive, tedious, and time consuming.

In our lab we have tested T7 protocol which works really well with the samples after MeDIP. That is why we can recommend this method for amplification.of MeDIP samples. Also from the feedback from our clients we know that WGA method is compatible with DNA samples obtained after MeDIP.

Diagenode recommends TLAD as a method for DNA amplification after IP. However we have previously used a Sigma WGA kit with our experiments before hybridizing on an array; is this method compatible with your MagMeDIP kit?
It is compatible, please see answer to the question above.
Spike control will be amplified by WGA2 kit (Sigma)?
Spike control as well as DNA of interest will be amplified. Spike control it is no mammalian DNA and it does not interfere with microarray.
What amount of DNA do you need to start with for DNA amplification?
It is depend on the amplification method you use. For T7 you can start even with 10-15ng per reaction. For other methods – please check the producer information.

Downstream applications: microarray, sequencing

After ChIP or MeDIP using magnetic beads, is there any step where I can store the DNA bound to the magnetic beads (before DNA purification with DNA purification module)?
Yes, knowing that it is not optimal condition that can result in DNA lost. DNA bound to the beads can be stored in last washing buffer.
I would like to use the IP’d DNA for hybridization on Methylation Array from Roche Nimblegen. They do recommend your Ab and kit as they prefer the magnetic beads. They have asked for around 3-6ug of IP’d DNA which would mean I am required to perform 30 reaction approx for 1 sample depending on the amount of IP’d DNA I obtain. So is there any way I can pool several MeDIP reactions at the start? Or Do you have any suggestions to make this more high throughput?

We do not recommend to pool down the samples at the beginning of the MeDIP protocol. Please, follow the protocol and use 1 µg of DNA per IP. The DNA recovery after MeDIP vary from 30 to 100 ng; microarray study usually requires 1-2 µg of DNA. Having your IP´d DNA you have several possibilities:

  1. to pool down several IP´d DNA samples
  2. to amplify your IP´d DNA using T7-Based Linear Amplification method.
  3. Any other method of amplification can be used keeping in mind that other protocol can introduce a bias.
Is it possible to use the internal controls from the MeDIP kit for microarray analysis with any commercial microarrays?
Currently, no microarray manufacturer has included the Diagenode internal controls.
For methylation microarray purpose, which protocol for DNA elution and purification is recommended?

DNA samples immunoprecipitated using MeDIP or MagMeDIP kit can be used successfully for microarray analysis. An average recovery after MeDIP (starting material is 1 µg) is about 20-50 ng of single-stranded DNA. As microarray study requires 1-2 µg of DNA, immunoprecipitated DNA should be amplified.

If you work with MagMeDIP (cat # mc-magme A10 or A32), DNA isolated with DIB buffer can be used directly for qPCR but for other applications it is better to not use DIB buffer, but to perform additional purification: elution with our DNA purification module (cat # mc-magme-002) with following phenol-chloroform extraction or columns purification. Note anyway that columns purification results in significant DNA lost (about 30%).

What is the anticipated yield of DNA per reaction? Will it be suitable for subsequent sequencing?
An average yield is about 20-50 ng and this is sufficient for next-generation sequencing. Note that recovered DNA is in single-stranded conformation and probably some optimization will be required for library creation for sequencing. Diagenode proposes the protocol for MeDIP-seq (please, look at the question/answer below), according to which the adaptors have to be ligated to DNA BEFORE MeDIP what will allow to use Illumina protocol.
What MeDIP-Seq protocol recommendations do you recommend?

The main steps for MeDIP-seq are quite simple but there are some variations. Diagenode has discussed the MeDIP-seq protocol with our expert customers. The following describes conditions tested by our customers for highthroughput sequencing using an Illumina system.

  1. Diagenode recommends sonicating the DNA so that the peak of material is quite short (150-250 bp); using the Diagenode Bioruptor® Sonicator, gDNA can be sonicated using the following protocol: Total time: around 40-50 min, Cycles: 30s ON, 30s OFF, Power settings: LOW Tubes: 0.65 ml silinonized tubes, Costar 3206 Volume in the tubes: 100µl Cooling sustem is recommended in order to get less sample-to sample variation.
  2. Diagenode recommends following the Illumina protocol for library preparation. Specifically, complete the end repair, A-tailing, and adaptor ligation. Some adaptations are detailed in Quail et al (2008) Nature Methods, 5 (12), pp1005-1010, which may be worth considering.
  3. Following library preparation (and clean-up) quantify the library using a DNA measurement device such as a Nanodrop or equivalent. Because the library's concentration is elevated due to the presence of 2x30bp adaptors, our customer’s recommend adjusting the concentration as follows (Diagenode recommends you independently determine whether this step is necessary or not): e.g.: (average size of sample [bp] + sum of adapter size [bp]( / (average size of sample [bp]) = elevation constant (175 + 60) / (175) = 1.343 100 ng/µl / 1.343 = 74.47 ng/µl Subtract 10% from the above to account for self-ligated adaptors e.g.: 74.47 ng/µl - (74.47 ng/µl * 0.12) = 65.53 ng/µl
  4. Use this concentration to add 1.2 µg to the MeDIP incubation mix, which is then performed using the IP-Star and Diagenode's Auto MeDIP protocols.
  5. Perform QC using qPCR for methylated and unmethylated controls that were added during the preparation of the incubation mix. Following the qPCR, purify the MeDIP’d DNA using the Diagenode’s DNA purification Module (mc-magme-002) and Phenol / Chloroform / Isoamyl alcohol or IPure DNA purification protocol.
  6. Following the general DNA purification step, the correct size of DNA of the input DNA and ‘MeDIP’d’ DNA is selected by excising DNA bands (50-350 bp) from a low melting point agarose gel. After clean-up of the size-fractionated DNA from the agarose gel, perform the adaptor-modified PCR. An alternate approach is to perform the adaptor-modified PCR, followed by the size exclusion on the agarose gel. The potential advantage is the higher concentration of DNA obtained after PCR. However a bias may be introduced as different insert sizes amplify at different efficiencies and this should be verified before modifying the recommended sequencing protocols.
  7. Following clean-up the MeDIP is ready to be sequenced
I would like to use DNA samples obtained with Diagenode´s MeDIP kit with the Illumina sequencer, but my samples need to be dsDNA. I could use the T7-Based Linear Amplification of DNA protocol but we will like to avoid any amplification as one of the advantages of the Illumina seq technique is to use very small quantities of DNA. How to obtain dsDNA without the use of any adaptator?

Concerning your question, there are 3 possibilities:

  1. As you know after MeDIP immunoprecipitated DNA is in single-stranded confirmation. If you use the T7-Based Linear Amplification of DNA you will get dsDNA.
  2. it is possible also to ligate the adaptor to DNA BEFORE MeDIP what will allow to use Illumina protocol.(the protocole for MeDIP-seq, please look at the question above)
  3. to get methylated fragments of DNA is it possible to use another method - MBD (instead of MeDIP). Using this technique you end-up with dsDNA:
    http://www.diagenode.com/media/documents/H6-GST-MBD_Sheet_1208V2.pdf