Nanodrop vs BioAnalyzer

Use this category for the exchange of ideas, methodologies and references regarding the isolation, manipulation and analysis of RNA. (Extraction protocols, Northern Blot analysis, RNase Protection, Differential Display, In Vitro Transcription, etc.)

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Nanodrop vs BioAnalyzer

Postby aazar » Sep 05 2017 1:21 pm

I ran an InVitro Transcription (IVT) reaction to make RNA for a protocol I'm using. After IVT I used Ampure beads to purify the reaction. I then quantified my RNA using the Nanodrop and it showed a concentration of 100ng/ul. When I ran the sample on a NanoChip on the bioanalyzer, I got a blank trace. I assume this means I only have free nucleotides in my sample but I'm a little perplexed because I thought that after a bead clean up I would have removed all free nucleotides. Any ideas why I don't see RNA on my NanoChip and why I didn't clear free nucleotides?
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Re: Nanodrop vs BioAnalyzer

Postby 29yrsExperience » Sep 11 2017 12:36 pm

It would be helpful to know what you are using for the IVT reaction. If you are using a kit, did you do the control template and did you get anything from that? If you are using a plasmid as template, was it linearized properly, and could there have been any RNAse A carryover from the plasmid preparation? Did you do a DNAse treatment after transcription to get rid of any leftover template? Are all your reagents RNase-free? Is your kit optimized for the size of transcript you are trying to make?

I’d review your kit instructions, or if you are not using a kit, look up some kit manuals on-line and read those because they have a lot of good troubleshooting tips and recommendations. Good luck.
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Re: Nanodrop vs BioAnalyzer

Postby DrShabba » Sep 22 2017 3:06 am

I would be careful with any nanodrop quantification of IVTs, they can be a log out (no joke).
Agree with 29yrsexperience...an all counts. Your RNA could be completely degraded, or the reaction hasn't worked properly (non-linearised plasmids would give a load of junk).

It would be helpful to know what your starting material was, as well as the kit you've used. I know the ambion kits come with a positive control for IVT synthesis, also the kits are very robust and give great yields.
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