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Old 07-17-2015, 05:39 AM   #1
Koncarius
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Location: Stockholm

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Default Possibility to run DNA/RNA duplexes on bioanalyzer

Hello forumers,
I am currently trying to find out if it's possible to run a bioanalyzer (Agilent 2100) on samples containing DNA/RNA duplexes.

So I have performed a cDNA synthesis on some rRNA and now I want to measure the size of the cDNA/rRNA hybrid fragments. I guess if I would use the RNA kit it would be impossible to distinguish free rRNA in my sample from the DNA/RNA fragments, but what if I use a DNA kit? I shouldn't have any DNA before the cDNA synthesis so in theory that should give me some information about the DNA/RNA hybrids right?

Or do you think I should do a second strand synthesis of my DNA/RNA (get rid of the RNA and replace with DNA) in order to get dsDNA and then use a DNA kit for analyzing?
I'm not very familiar with the exact chemistry behind the dyes of the different bioanalyzer kits and I had a hard time to find this information on the web.

Thank you in advance
/Koncarius
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Old 07-17-2015, 04:10 PM   #2
kerplunk412
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Location: Bioo Scientific, Austin, TX, USA

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Default

I would recommend doing the second strand synthesis, purifying, and measuring that product on a DNA chip. Even if the DNA/RNA hybrid shows up on the DNA chip it might not run at the expected size, and I think having a bunch or residual RNA may make the chip run weird or the data difficult to interpret, even on a DNA chip. Also, if your eventual goal is library prep it makes more sense to check the double-stranded cDNA, as this will be the inserts in your library.
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