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Another problem regarding qRT-PCR/DNase treatment

dnase real time SYBR green RNA pcr

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#1 kutayozturk



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Posted 29 May 2012 - 05:31 AM

I want to make sure of everything before starting, so after DNase treatment, I have checked them in qRT-PCR, using the same cycles that I use in my relative gene expression analysis. This way I could make sure that DNase treatment works efficiently, no residual DNA left. It seems that when I treat them with DNase, I see a significant difference between it and non-treated RNA samples. However, I see that all treated samples as well as negative control start to give flourescence at around Ct-35, and amplify a little bit till Ct-40. When I look into dissociation curve, I see the same small peak in all the samples treated with DNase. I have done this experiment also to check if my newly ordered primers are fine. I will mark them in one of the pictures, so if you can help me if there is a problem with the primer, or anything else, I would appreciate.
ps. Even more surprisingly, I see that in positive control and non-treated samples amplified with a primer, I see no amplification at all (Probably due to insufficient mix of primer before dilution), however I see them giving flourescence in treated and negative control samples. Is there any possibility that my SYBR green mix is contaminated somehow? This is only the 3rd time I used this tube and in fact in all of them I had problems. I am putting some the images as attachment

If theres is anything else you want to know, please inform me

Attached Thumbnails

  • dnase treatment amp plot.jpg
  • dnase treatment dissociation curve.jpg
  • negative control dissociation curve.jpg
  • Negative control_amp plot.jpg
  • non-working primer amp curve.jpg
  • non-working primer diss curve.jpg
  • overall amplification plot.jpg
  • overall dissociation curve.jpg

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