ChIP on chip Affymetrix - Ampification protocol - Quality of amplified DNA (May/10/2007 )

Dear all,
I make ChIP on chip experiments on Affymetrix tiling arrays. I used the Affymetrix protocol to amplify my ChIPed DNA, using dNTPs + dUTP.
The nice thing is that I still see the enrichment of my positiv targets after amplification.
However - when I use 1 ng of my Chiped DNA as template, I get signal after 28 cycles, but when I use 1ng of my amplified DNA, I get a signal after 32 cycles. I am afraid that I get some kind of artefact during amplification, whoever this might also be due to the UTP in the amplified sample.

Has anyone ever had similar problems?

Thanks, Fridjtof

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Hey did you ever figure it out?

I'm having a problem with simply amplifying my DNA at all. If you get a chance, would you tell me your protocol for amplification. I'm following the affy protocol, adding a little bit more Mg and about 5 extra cycles.
Thanks.

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Hi C,
No, but i got a little further, my problems are due to some contamination in the chip´ed DNA. Amplification of e.g. the diluted total input works absolutly perfect.

I also follow the Affymetrix protocol.
When you get no product at all, I can only think of two reasons. Maybe your polymerase doesn´t work with dUTPs, or your concentration of chip´ed DNA is to low. Have you measured the conc. of your DNA? And have you ever tried the amplification protocol with diluted total input or simply some genomic DNA?

Good Polymerases for dUTP amplification are THERMOPRIME PLUS from ABgene or ThermoPol from NEB.

Let me know about your progress,

Best wishes,

Fridtjof

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Hi Fridtof,
Did you figure out the answer of your question? I have a very similar problem. I had a consistent Ct values after IP using a real-time PCR. However, amplified DNAs did not. Please share with me any finding.
Thanks,

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Hi,

I´m sorry for the late reply, I hope you will still find this.
I was able to figure out, but my problem was very "special". I have not prepared the chip-samples myself, and the TA who has prepared them has used hering sperm DNA to block unspecific DNA binding ! - so my artefact is amplified hering sperm DNA. I haven´t known this - cost me 3 month of useless work to find out.

However, I prepared new samples, an tried different amplification protocols, mainly variations of the affymetrix protocol and the WGA-Kit from Sigma.
I am not sure if I really understood your problem.
Do you get inconsistent ct values on the same amplified DNAs, or is there an inconsistency between different amp reactions?
There is allways some variation after amplification. I got this down to 3-4 ct, meaning a 10-fold change after amplifcation.
I´m not sure if this can be further improved, I will now use these samples for hybridization. My positiv controls are 40-fold enriched, so they should be visible over the noise.
Please, tell me what exactly your problem is, maybe i can help.


Hi Fridtof,
Did you figure out the answer of your question? I have a very similar problem. I had a consistent Ct values after IP using a real-time PCR. However, amplified DNAs did not. Please share with me any finding.
Thanks,
[/quote]

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hi everybody, I am new to microarray.I just wanted to have a feedback about using ChIp on chip protocol from affymatrix.Like I had a doubt regarding biased amplification from random primers which seems actually true after read this forum topics.I have other many doubts and seek answers to those

1.why same seq of random primers(except the N9 overhang)is used for both ronds of amplifications.could not we reduce biased amplifications using two different sets of random primers............? OR may be i am totally mistaken the basic of amplification.

2.they dont mention starting amt of template for PCR, its just a 10 ul vol but no ug mentioned it confuses me a bit.

Please help me understand the very basic of microarray.

Thanks.

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