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problems when use qPCR to quantify bisulfite modified DNA


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4 replies to this topic

#1 LeiLi

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Posted 19 July 2010 - 01:11 PM

I use EpiTect kit to do the bisulfite conversion and use qPCR to do the quantitation. But the results do not match with the amount of the DNA I use in the conversion. For example, I use 2 ug of genomic DNA (measured by qPCR, a well established method in our lab) for bisulfite conversion and after the conversion, I performed another qPCR designed for bisulfite converted DNA, and it always seems to have more than 2 ug DNA in it. I tried different std curves and vary primer concentrations or DNA templates, and could not fix it.

I am wondering whether anybody else has seen the same problem. Or what else I can try to fix it. I need to know the accurate DNA concentration!!

Thanks a lot!

#2 criscastells

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Posted 20 July 2010 - 04:50 AM

I use EpiTect kit too but I have never had this problem. Normally I have only a 20% of the genomic DNA. Do you wash correctly the converted DNA? There could be some interference if the converted DNA is not correctly washed. I do not use qPCR for the quantification, but I supose it is the same as spectrophotometric measurements.

I hope it can help you a little

#3 LeiLi

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Posted 20 July 2010 - 06:47 AM

I just followed the protocol to do the wash. I did not use carrier RNA, since the amount of DNA I used in the conversion is good.
I am not sure how accurate the nanaodrop can measure the concentration.

#4 ElHo

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Posted 22 July 2010 - 06:30 AM

Maybe the values of your standard curve are wrong and thatīs why you get those strange results. From your description I conclude that you perform an absolute quantification running a standrad curve of known concentrations in parallel with your samples. I just wonder how you prepared the DNA for your standard curve and determined the concentrations. Because as far as I know itīs not that easy to measure the concentration of converted DNA because itīs partially single-stranded. So spectrophotometry will not work.

#5 LeiLi

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Posted 22 July 2010 - 09:48 AM

I think you are right. I just used the control DNA also purchased from Qiagen, Epitect control DNA to do the std curve. Although they claimed the concentration is 10 ng/ul. I tried several methods to quantify it and did not get 10 at all. But I have to trust it,'cause like you said, it is hard to quantify DNA after conversion. I have to have a starting point.

Maybe the values of your standard curve are wrong and thatīs why you get those strange results. From your description I conclude that you perform an absolute quantification running a standrad curve of known concentrations in parallel with your samples. I just wonder how you prepared the DNA for your standard curve and determined the concentrations. Because as far as I know itīs not that easy to measure the concentration of converted DNA because itīs partially single-stranded. So spectrophotometry will not work.






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