bioanalyzer - details

[LukaszKielpinski]

Hi Everybody!

Most of us use Bioanalyzer Chips to check the libraries (and RNA if working with it) but unfortunately Agilent website is not mentioning much details about how does this appliance work.

I am extremely interested (due to strange results) what is the denaturation method applied for RNA sample? is there urea (doubtfully) included? or is it maybe only very high temperature in low ionic conditions?

Another question (less important) is what dye is used to stain nucleic acids for detection?

If any of you was able to find answers to this simple questions I would really appreciate sharing this knowledge.

Lukasz

[colicoli]

The Bioanalyzer runs at 42 degrees C. Check it, you know it's not 65 or the chip would be hot at the end. The buffer includes a denaturant but it is not completely denaturing. If you run oligos or small RNAs with known secondary structure, you'll see patterns consistent with partial melting. I'm not sure of the dye but there are different ones for the DNA and RNA kits, so you may see different effects with mixtures of ss and duplex. For typical complex mixtures of RNA, none of this matters as it is pretty reproducible. If you are comparing an RNA with specific structure to denaturing PAGE gels, be sure to run controls on the same chip so you know what to look for. Good luck!

[mikaelk]

As far as I know, the samples should be heat denatured (2mn at 70°C) prior to loading on the chip.
Once denatured, the samples are kept on ice until loading. This is what is specified in the user guide.
The buffers and running temperatures are just there to limit the secondary structures development after loading but not more.