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cell free DNA and maintaining stability

DNA extraction cell free DNA

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3 replies to this topic

#1 why_not_wheat

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Posted 28 April 2013 - 05:47 PM

Hey,
I'm looking to start examining cell free DNA and I'm worried about it's purported short half life.

Can anyone give me a working estimate on it's half life/stability??

Has anyone used reagents like Invitrogen's RNALater or Qiagen's Allprotect tissue reagent, to maintain the integrity of cfDNA for longer periods of time?

thanks!!

#2 phage434

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Posted 29 April 2013 - 04:53 AM

DNA is very very stable at slightly alkaline pH and in the absence of magnesium. TE is designed to store DNA for long periods, and DNA stored in TE is stable even at room temperature. RNA is an entirely different matter.

#3 why_not_wheat

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Posted 29 April 2013 - 01:38 PM

Thanks phage434, I know that's true for genomic/plasmid DNA, but for circulating cell free DNA the literature is stating it has a half life of between 15-30min. Longevity of the cfDNA is apparently maintained with -20 and -80 freezing, which I won't have immediate access to as I collect my samples, and I don't think I will be able to change my sampling protocol (bit of a catch22 situation).

So that's why I'm curious about the RNALater and Allprotect products.

#4 phage434

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Posted 29 April 2013 - 02:02 PM

DNA is a chemical. It's the same whether it is in a cell or not, although the cell provides some physical protection for long strands. cDNA is single stranded, which may be somewhat less stable than double stranded DNA, but it is still very stable under the right circumstances (in TE, for example). You need enough TE to chelate the magnesium from the reverse transcription buffer (probably at least 10x the volume of TE than the buffer, since the buffer is likely 10 mM MgCl2 and the TE is 1 mM EDTA).





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