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ChIP-qPCR gives odd amplification plot


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#1 mhong

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Posted 02 August 2011 - 09:50 AM

Hi all,
I was hoping for some help with a problem analyzing my ChIP-qPCR. My amplification curves are shaped oddly and sometimes sigmoidal. When I look at raw fluorescence, I see that it starts to come up above background in early cycles, levels off, and comes up again. When I run a gel on the product, I only see my intended amplicon.

I think it may have to do with my fragment size (200-600bp), but am not sure what I can do about it during analysis. I've been trying to fiddle with the baseline settings, but have not been terribly successful. Any advice would be appreciated!

Attached Thumbnails

  • amp plot.png
  • raw fluorescence.png


#2 chabraha

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Posted 02 August 2011 - 10:40 AM

you amplicons may be too big, mine are no more than 150 and most are ~75bp..........also, SYBR green wont discriminate between intial DNA and amplified DNA, so you may need to back off a bit in the DNA content. It could also be mispriming in the initial few rounds of PCR
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#3 mhong

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Posted 02 August 2011 - 02:28 PM

Chabra, Thanks for the tip! My amplicons are sized between 60-110 bp, most around 65. I am also loading 1ng to a 5ul reaction. Do you think this is still too much? I checked the primers using 5ng of genomic DNA and get Ct's about 7 cycles lower. Do you think there's anything I can do at this point to make my data analyzable?

you amplicons may be too big, mine are no more than 150 and most are ~75bp..........also, SYBR green wont discriminate between intial DNA and amplified DNA, so you may need to back off a bit in the DNA content. It could also be mispriming in the initial few rounds of PCR



#4 chabraha

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Posted 02 August 2011 - 06:38 PM

What samples are these that give you the sigmoidal curve? I assume that you run all your primer sets on the same plate....do they all have the same efficiencies for the given conditions?..........I like to have my Cts between 18&30 so maybe still back off a bit on the DNA amount......how exactly are you measuring your ChIP'ed DNA? Nanodrop? If so I wouldn't trust your measurements. Try running 10fold serial dilutions of your input DNA......see if its a primer problem.....this is my suspicion since the rest of your real-time rxns look ok. If you want to still use this data raise the threshold for your background.
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#5 mhong

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Posted 08 August 2011 - 10:25 AM

Thanks again, chabra

Its just one of the two ChIP samples that gives sigmoidal curves on a small subset of primers.

The samples I'm using are actually amplified libraries made for ChIP-SEQ and were quantitated using the Qubit fluorescence reader. What I'm trying to accomplish here is validation of the sequencing results. I ran the primers on my input sample and genomic DNA as you suggested and it gives normal looking amplification curves.

I'm worried that my fragments are smaller than I think and my amplicons are too big for them. Would this cause the results I've seen?

I made all my primers with PerlPrimer using the same parameters. I designed them specific to binding sites and neighboring non-binding sites so that I could validate the sequencing results. I guess the biggest problem is that my qPCR data does not validate the ChIP-SEQ! The input shows enrichment of binding regions in some cases, but not in others.

Edited by mhong, 08 August 2011 - 12:33 PM.


#6 chabraha

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Posted 09 August 2011 - 02:19 PM

Ah, ok.....its definitely possible.........run your library out on a gel and look at the size of the smear.......and don't load to much DNA in the gel........doing this can lead to overestimation of sheared DNA size. How are you checking your sheared DNA size before doing the IP?
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