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ChIP PCR question


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#1 krystle

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Posted 22 February 2011 - 01:39 AM

Hi, I've got my chip samples and i'm ready to do some PCR. But before i perform the pcr, i'll just like to check if theres anything that i should take note for the PCR conditions. I thought I should run all my primers with the input to make sure that it works. But how much of the input should i use to run the PCR? Is there a standard amount that i should use? I saw somewhere else that I might need to dilute the input 10X to 80X before i run pcr? Whats the reason for that? And if i'm doing normal pcr, do i stick to a standard number of cycles, as in real time PCR which is 40 cycles? Or do i need to optimise and vary the number of cycles?

And for subsequent PCR using the IP samples, do i need to use the same volume or same amount (determined by nanodrop)? Is there a difference in the amount of samples that i should use for normal PCR vs real time pcr?

Please let me know also if there is anything else that i should take note of.

Sorry for the many questions.

#2 chabraha

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Posted 22 February 2011 - 09:11 AM

How much DNA to use depends on your extraction method, Chelex tends to give less total DNA recovery than traditional elution methods. I use the chelex method and elute my DNA in 200ul and use 9ul per RT-PCR. If your not going to use real time PCR I would shoot for 20-25 cycles, this would hopefully get you in the liner range. As for PCRing of your IPed samples, you need to run the same volume, not DNA amount.
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#3 krystle

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Posted 22 February 2011 - 02:13 PM

How much DNA to use depends on your extraction method, Chelex tends to give less total DNA recovery than traditional elution methods. I use the chelex method and elute my DNA in 200ul and use 9ul per RT-PCR. If your not going to use real time PCR I would shoot for 20-25 cycles, this would hopefully get you in the liner range. As for PCRing of your IPed samples, you need to run the same volume, not DNA amount.


Hi. How do I know for sure that my sample is in the linear range? Is there a way to check? Maybe serial dilution of the sample? And why is it same volume of ip sample? Dont i need to check and Ensure that the ip pcr is also in linear range? How do I show my data after the pcr? I thought it should be band or ct of ip sample using primer of interest divided by that of same sample with neg ctrl primer. And this value should be higher then that of those done with mock ip sample. If tts the case then how does the pcr using input comes in?

If I worked with abt 25ug sample per ip, aliquoted 1/100 volume out as input, does it means I have 100x more chipped sample than input if same volume were used for pcr?

Thanks .

#4 Mighty Mouse

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Posted 22 February 2011 - 03:45 PM

If I were you I would try optimizing your PCR using some genomic DNA from the organism of interest instead of wasting your more precious ChIP DNA. I've never done end-point PCR for evaluating ChIP, but I don't think you can really get much quantification out of it, at least I don't think I've come across anyone doing that in the literature (but I wouldn't be surprised if someone has done this somewhere). Typically if you are using end-point PCR it is considered semi-quantitative at best, so you better have a big effect!

I believe to determine your linear range for your PCR you would want to first run out on a gel your PCR product after stopping at various cycles in the reaction. The idea is to make sure you don't have a saturated signal due to being in the plateau phase of the PCR run, you want to be in the exponential phase, the same place in which you would determine your Ct value from a qPCR run. I think you can more or less do this by eye (i.e, this is all only semi-quantitative anyway). It might not hurt to run some serial dilutions of your DNA as well, but I would be careful not to have dilutions that are too many orders of magnitude different from your 1% input (perhaps to 1:2 dilutions instead of 1:10). This will also give you some idea as to how sensitive your assay really is.

Since you are not running qPCR, your input sample functions primarily to demonstrate that you have DNA and to give some idea as to how much DNA was successfully ChIP'ed (relative to Input). You run equal volumes of DNA after your ChIP so you can compare whatever your experimental treatments are and compare your ChIP to your mock and input. If you used varying volumes you wouldn't really be able to make any meaningful comparisons.

As far as setting up so your input and IP samples are in the linear range, the most important thing is ensuring that your IP samples are in the linear range (because you are not making comparisons among the input, that doesn't tell you anything). However, in my experience ChIPing for various transcription factors, your ChIP sample is typically around 1% of your input. For histones and histone modifications this would probably be quite a bit higher. But if you are in the linear range of your PCR you are probably fine as the linear range probably spans about 5-10 or so cycles.

As for presentation, if you are running end-point PCR, you would just present the visualized bands, like a western blot. You can't calculate a Ct using this method.

Hope that helps.

MM
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#5 krystle

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Posted 22 February 2011 - 04:00 PM

If I were you I would try optimizing your PCR using some genomic DNA from the organism of interest instead of wasting your more precious ChIP DNA. I've never done end-point PCR for evaluating ChIP, but I don't think you can really get much quantification out of it, at least I don't think I've come across anyone doing that in the literature (but I wouldn't be surprised if someone has done this somewhere). Typically if you are using end-point PCR it is considered semi-quantitative at best, so you better have a big effect!

I believe to determine your linear range for your PCR you would want to first run out on a gel your PCR product after stopping at various cycles in the reaction. The idea is to make sure you don't have a saturated signal due to being in the plateau phase of the PCR run, you want to be in the exponential phase, the same place in which you would determine your Ct value from a qPCR run. I think you can more or less do this by eye (i.e, this is all only semi-quantitative anyway). It might not hurt to run some serial dilutions of your DNA as well, but I would be careful not to have dilutions that are too many orders of magnitude different from your 1% input (perhaps to 1:2 dilutions instead of 1:10). This will also give you some idea as to how sensitive your assay really is.

Since you are not running qPCR, your input sample functions primarily to demonstrate that you have DNA and to give some idea as to how much DNA was successfully ChIP'ed (relative to Input). You run equal volumes of DNA after your ChIP so you can compare whatever your experimental treatments are and compare your ChIP to your mock and input. If you used varying volumes you wouldn't really be able to make any meaningful comparisons.

As far as setting up so your input and IP samples are in the linear range, the most important thing is ensuring that your IP samples are in the linear range (because you are not making comparisons among the input, that doesn't tell you anything). However, in my experience ChIPing for various transcription factors, your ChIP sample is typically around 1% of your input. For histones and histone modifications this would probably be quite a bit higher. But if you are in the linear range of your PCR you are probably fine as the linear range probably spans about 5-10 or so cycles.

As for presentation, if you are running end-point PCR, you would just present the visualized bands, like a western blot. You can't calculate a Ct using this method.

Hope that helps.

MM


Thanks for your reply. Does it mean that if i were to do real time pcr i won't have to go through the trouble of determining the linear range of amplification? It seems like analysis using real time PCR is much more straight forward? So how much sample should i try for a start if i want to do real time pcr? I think because i was following someone else protocol so my stuff if a bit different. I used 120ul (25ug) of sample to chip. And i had 30ul of the sample saved as input also. That means my input is 25% right?

#6 krystle

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Posted 23 February 2011 - 12:15 AM

I did a trial real time pcr and this is what i get.

Using primer of interest,
Ab of interest IP: 31.86
His tag Ab IP (mock):32.85
10%input: 23.83

Using negative control primer,
Ab of interest IP: undetermined (>40)
His tag Ab IP (mock):undetermined (>40)
10%input: 23.53

I followed some methods online to calculte %input enrichment but it didn't quite make sense to me. Can someone help me with this? It seems like there is some enrichment at the region of interest but its very little? Like 2 fold? What should i do if I want to try and amplify this effect on PCR? Run real time pcr with more templates, or use more antibody to pull down?

Also, if i want to translate this to normal end-point PCR, does it mean that i should run PCR on my 10%input sample with around 23 cycles, and on my chipped samples with around 32 cycles? Am i making sense?

Also, I checked the melting curve of my primer. Although it is a single peak, the fluorescence level is very low, just 4-5 times above the background noise. Is it okay?

Edited by krystle, 23 February 2011 - 12:21 AM.


#7 Mighty Mouse

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Posted 23 February 2011 - 01:03 PM

I did a trial real time pcr and this is what i get.

Using primer of interest,
Ab of interest IP: 31.86
His tag Ab IP (mock):32.85
10%input: 23.83

Using negative control primer,
Ab of interest IP: undetermined (>40)
His tag Ab IP (mock):undetermined (>40)
10%input: 23.53

I followed some methods online to calculte %input enrichment but it didn't quite make sense to me. Can someone help me with this? It seems like there is some enrichment at the region of interest but its very little? Like 2 fold? What should i do if I want to try and amplify this effect on PCR? Run real time pcr with more templates, or use more antibody to pull down?

Also, if i want to translate this to normal end-point PCR, does it mean that i should run PCR on my 10%input sample with around 23 cycles, and on my chipped samples with around 32 cycles? Am i making sense?

Also, I checked the melting curve of my primer. Although it is a single peak, the fluorescence level is very low, just 4-5 times above the background noise. Is it okay?


Frankly, with all your questions it sounds like you need to take a step back and evaluate what exactly it is you are trying to do and think about how the technique can or cannot answer your research question.

With qPCR you can quantify your ChIP whereas you cannot really do this with end-point. Since I don't know what sort of questions you are asking it's hard to tell what the best route is for you to take. Whichever way you go there is a bit of work upfront that needs to be done. If you are doing qPCR you need to test primers for efficiency and specificity. If you are doing end point you need to get into the linear range and check primers for specificity, although this is not as much of an issue since you'll be running your products out on a gel.

As for your question regarding how my cycles to run end point, looking around 23 cycles would probably be a good place to start considering your qPCR data. You should run all your samples to the same number of cycles as you are not interested in making comparisons among your input samples...

Remember the comparison of most importance for you is not the antibody versus mock (this is a good qualitative control) but your factor binding to your region of interest versus a region that is not thought to bind your factor.

MM
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#8 krystle

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Posted 23 February 2011 - 02:16 PM

Hi mm, The ultimate thing tt I need to prove is that the tf i pulled down binds to the region of interest. Getting to know exactly how much more it binds as compared to other regions is not necessary, just a plus. That is why I thought I could make do with just end point pcr. But just seems like most people were doing real time pcr so I was wondering if I should do that as well, since it's not too difficult to do that either.

Hope you can advice me on this. Thanks so much

#9 chabraha

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Posted 23 February 2011 - 03:30 PM

Krystle,

I think that the point MM is trying to make is that you cannot interpret whether your TF of interest binds SIGNIFICANTLY without assaying its activity at a DNA region that you know your TF of interest does not bind. THe fact that your IP w/specific antibody is greater than the IP using a non-specific antibody demonstrates that one antibody binds your TF of interest with higher efficiency than your non-specific antibody, it means nothing more than this. If you can show that your specific antibody IPs your TF at your region of interest more than it binds at a region where you suspect your TF has no activity it means your TF is truly associated with your DNA sequence of interest. THe reason that you save a bit of the material to be IPed before you perform the ChIP serves not only to ensure your PCR is working but also have something to normalize your data to. From the looks of your initial real time run you need to add more DNA to the PCR reaction Ct values between 20-30 are best for reproducibility, anything lower and your pushing the limits of confidence for RT-PCR. For example, my 1% Inputs usually have Ct values ranging from 18-23, and my specific IP at my site of interest has Ct values 3-5 less than that of my 1% Input. Hope this helps
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#10 krystle

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Posted 23 February 2011 - 05:39 PM

Hi chabraha,

I know that I need to compare the result to a region that I dun think the tf binds, which is why I have the neg ctrl primer. And based on my first run, there is amplification when using primers that span my region of interest, but no amplification when looking at another region i dont think the tf binds. In other words, the tf truly bind to the sequence that I think it should bind right?
However, the ip done using another ab that is not supposed to bind to the tf of my interest also gave similar result, and I'm now wondering why is tt so. Or what can I do to figure this out. And of cause I'm also wondering supposedly I do clear that problem, is the data sufficient already to convince my prof that the tf binds to the region Im interested in. Thanks.

#11 chabraha

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Posted 24 February 2011 - 08:21 AM

After calculating your %Inputs I got .019% for your His-tag Ab and .038% for your TF Ab. So if you subtract out your non-specific IP then your left with .019% Input, which is not great but the antibody to your TF may simply not be as good as some other antibodies, maybe check the literature for ChIPs done on the same TF and see what other groups got. It is good that you don't see any signal using the negative control primers, however I have found that when your specific IPs are this low that reproducibility becomes an issue. In my experiences it is best to always do a control ChIP just to convince your boss (and yourself) that you can successfully pull off the experiment. Maybe choose a TF that has a good antibody available and has already been published on so you can just use already validated primer sets. Perform the experiment, and compare your results with those that are published. I would also (if you already haven't) validate your TF antibody, some aren't suitable for ChIP, but showing that your Ab can detect your TF by western blot or IF may provide some valuable insight as to the efficacy of the antibody. Lastly, because your non-specific IP was half of you specific IP I believe, and literature supports that your data is not acceptable for claiming true enrichment of your TF at your site of interest. Maybe EMSA would be a technique worth investigating? Just a thought, anyways hope this helps!
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#12 krystle

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Posted 24 February 2011 - 02:24 PM

Hi chabraha, thanks so much. Think I'll do a check of the ab to check their efficiency. Thanks so much again. By the way, can I just check a few more things. How much %input diff should I get to be a convincing enough number for enrichment? Is there a min number standard? Also, regarding the chip protocol, I preclear my sample using the relevant IgG isotype before incubating the actual conjugated ab with the precleared Sample. How important is it to preclear the sample? If I don't preclear, is it as good if I just do an additional ip using the isotype IgG for comparison? Thanks again

#13 chabraha

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Posted 25 February 2011 - 07:19 AM

Ideally I like to see at least 100 fold enrichment for my specific antibody IP over my IgG (non-specific) IP at my positive control region. That is not a hard and fast rule, and I'm not sure there is one out there but you can imagine what you'd believe if you were reviewing a paper with ChIP data, so I'd go with whatever would convince you the data is significant. Also, I do not pre-clear my ChIPed material. I just dilute my sample 1:100 in ChIP dilution buffer, add my antibody, add my beads and let them go overnight. I always include an IgG IP for non-specific interactions. Just out of curiosity how many cell equivalents are you using per IP?
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#14 krystle

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Posted 25 February 2011 - 07:48 AM

My protocols require me to dilute my sample 10x only. I use supposedly 25ug chromatin per ip. And if I back calculate that, it's about 1.25-1.5 million per ip. Is that too little/much? And I use 20ul beads for each ip.

100 fold over IgG? So my previous data is like only 2 fold right? Oh my god. 100 fold sounds impossible to attain. :x

#15 chabraha

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Posted 25 February 2011 - 08:15 AM

Sorry I meant I dilute 1:10, just like you. I'd day your in the right ballpark for cell numbers I use ~2x10^6 cells per IP with 20ul Protein A magnetic beads. How about your elution method? 100 fold doesn't need to be reached by an means, remember, the antibodies your using are vastly different than the ones I'm using that's why its important to try and identify a positive control region where you know you TF of interest binds so you can truly be confident your IP and antibody are working effectively. Just a thought but you may want to try your IP with antibody alone overnight and then add in the beads for an hour or so or just add them together and let them go overnight,mix it up a bit and see if you can improve your IP efficiency. If you can't maybe try another antibody. Also, as long as your specific IP is pulling down your TF with statistical significance over your IgG IP you'd probably be ok.
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