WGA problems CpG island amplification - (Jan/21/2008 )
Another problem I am having with Sigma WGA II protocol. I notice that the enrichment peaks in my chip-on-chip tend to be non-existent in my CpG islands. I know lots of PCR and sequencing problems arise in GC-rich DNA, so it makes sense there might be limitations in WGA for these regions. Has anyone else encountered this and if so has anyone tried adding any PCR enhancers to the WGA protocol (eg DMSO, betadine)??
we have noticed this too with our affy promoter arrays
what arrays are you using?
personally im leaning towards it being a array platform problem as opposed to an amplification problem, as after WGA we can still see enrichment within the CpG island via qPCR.
Hi:
We are also using Sigma WGA, but in the qPCR, when we try to make a standard curve on WGA Input, it looks terrible compared to non-amplyfied Input. (curves showing up 4 cycles later, bad replicates, higher concentrations not amplyfying over the threshold).
We are hesitant to hybridize on an array having such bad results on the real-time.
Does anyone have some idea why this happens? (Inhibitors, linker associated disruptions???)
Help.
Thank you
Diana
We are also using Sigma WGA, but in the qPCR, when we try to make a standard curve on WGA Input, it looks terrible compared to non-amplyfied Input. (curves showing up 4 cycles later, bad replicates, higher concentrations not amplyfying over the threshold).
We are hesitant to hybridize on an array having such bad results on the real-time.
Does anyone have some idea why this happens? (Inhibitors, linker associated disruptions???)
Help.
Thank you
Diana
Hi Diana,
I have also noticed the same thing and did start a thread on here before xmas but it didn't really go anywhere :/ Are you doing the suggested 10ng for 14 cycles? I have spoken to other people who routinely do chip-chip and they don't even qPCR their amplified material before hybridisation (because they always got wierd results). They went on to hyb however and got good results. I have got ok standards curves after WGA and went on to hyb and it looked reasonable, but I did stuff up a few things along the way (it was my first hyb). I'll have to see how the next hyb goes
Hi,
I have used Sigma WGA method for Chip on chip arrays..eventhough the amplifcation showed weird patterns, the microarrays worked well. I use Agilent microarrays.
I find really nice results with the WGA on my arrays outside of CpG islands. I have not tried qPCR from amplified material, but rather from the original IP samples just to verify that the trends I am seeing in the microarray are similar to the original samples. I am intrigued by possibility of this being an issue with the array hybridization its self. I am using agilent arrays, 60mers probes adjusted to a similar Tm. I wonder if there is an issue in the lack of complexity in the CpG island DNA that contributes to more background noise in these regions?
We are also using Sigma WGA, but in the qPCR, when we try to make a standard curve on WGA Input, it looks terrible compared to non-amplyfied Input. (curves showing up 4 cycles later, bad replicates, higher concentrations not amplyfying over the threshold).
We are hesitant to hybridize on an array having such bad results on the real-time.
Does anyone have some idea why this happens? (Inhibitors, linker associated disruptions???)
Help.
Thank you
Diana
could you post a standard curve for us to have a look at?
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