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Strange looking "elevated" amplification plots from ChIP-qPCR samples


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#1 CellBiologist

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Posted 27 January 2013 - 06:51 AM

Hi everyone,
I am hoping that you all can help as I am not an expert in qPCR. I performed a ChIP experiment to assess binding to human ribosomal gene repeats. I used primers that have been published in multiple papers and a positive ChIP control: UBF. I have attached the strange amplification plots that I get when doing a standard curve using a dilution series of 1:10 of the input ChIP sample. I have used this same input sample for other ribosomal gene repeat primers and I get a "normal" looking amplification curve. It looks as though the qPCR reaction does not go into the log phase until many cycles into the PCR; the threshold also looks to be much more elevated than most reactions. Can anyone provide some insight as to what these plots mean? I greatly appreciate any and all help you could provide.
Thank you,
CellBiologist

Attached Thumbnails

  • amp_plot_01.jpg
  • amp_plot_01zoom.jpg


#2 Lisa Hsu

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Posted 05 February 2013 - 05:11 AM

Will there be a problem with the reagent you've used ?
Is this SYBR green ?
I get this similar looking graph when SYBR green has gone off.
Just a thought ....

#3 jerryshelly1

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Posted 06 February 2013 - 09:27 AM

Higher threshold may be due to you having fewer gene products after completion of your ChIP. During normal RT-PCR, cDNA production from RNA produces a greater number of gene products that can be amplified by your primers. This allows your Sybr green detection from DNA incorporation to exceed your background Sybr green background reading more quickly (giving you a lower threshold value).

Maybe your ChIP antibody is not very specific, causing a overall decrease in ChIP gene products. What does your input sample look like? Do the input Ct values appear lower, as compared to your ChIP Ct samples?

Maybe increase Ab concentration, Cell number, bead number (depending on affinity).

Hope this helps




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