DNA and salt contamination using mirVANa miRNA isolation kit - (Mar/08/2010 )

Hi everyone, I need some help on this matter. I'm extracting miRNA using Ambion's mirVaNa miRNA isolation kit. I got pretty poor quality results. My agarose gel has lots of smearing at the top of the gel, which i believe is caused by DNA and salt contamination. Does anyone has a DNA removal step protocol using lithium chloride precipitation? I had surfed the net for it. There's a lot of variation in the lithium chloride protocols. My samples are cancer cell lines from cell culture. I'm trying to avoid DNAse treatment, as the heat inactivation step can damage RNA. Also, can anyone recommend a salt removal protocol?

I believed that the poor quality of my samples is due to the evaporation of my wash solution 1 and 2. It's dissolved in 100% ethanol. I think i left it out in the open for too long. What do you think would happen if i tried topping up the wash solutions with more ethanol?

Drop dialysis is an easy and quick way to remove salt from samples. You can make the wash (buffer PE, or similar) fresh with the recipe here:
http://openwetware.org/wiki/Qiagen_Buffers
It's just Tris HCl pH 7.5 10 mM, with 80% ethanol.

Just to confirm.. Wash solution 1 is Tris HCl pH 7.5 10mM with 80% ethanol. So both Qiagen and mirVana wash solutions are the same? What about wash solution 2?

phage434 on Mar 8 2010, 09:31 PM said:

I don't know what the mirvana washes are. They are probably quite similar, however.

Hi Kaluts, I believe your guess about the old buffers are true. I had the same problem once and I just used the Qiagen RPE buffer instead of WB2/3 of the mirvana kit and it worked perfect. Not sure of the substitute for the miRNA wash solution. But the product manual says it contains guanidium salt!! I'm wondering why!

Hope this helps

Thanks for clearing my doubts. I'll try it out. But does anyone has a protocol for removing DNA contamination using lithium chloride?

gogreen on Mar 11 2010, 12:39 AM said:

Hi Kaluts, I believe your guess about the old buffers are true. I had the same problem once and I just used the Qiagen RPE buffer instead of WB2/3 of the mirvana kit and it worked perfect. Not sure of the substitute for the miRNA wash solution. But the product manual says it contains guanidium salt!! I'm wondering why!

Hope this helps

Hi - For DNA removal use an on-column DNase digestion - this is the only way to get rid of all the DNA from your sample. Try the Qiagen DNase as this does not require heat inactivation. You could probably incorporate the qiagen DNase into your current kit and wash it away during the wash steps rather than heat deactivate. I use the miRNeasy extraction kits (Qiagen) which has an optional DNase step that does not require heat deactivation. If you already have the RNA isolated and have no more sample you could also put the RNA back through the column (you will lose some sample but you will at least get rid of the DNA!). MiRneasy Micro can be used to isolate small amounts of 'small' RNA and we now use it instead of the mirvana kit. I would also recommend analyzing your sample on a bioanalyzer pico or nano chip (depending on the amount of RNA you have). It only uses 1ul sample and will give you a much better representation of your RNA integrity +/- Dnase digestion.
I hope this helps!
PS p/c based extractions never get rid of all the DNA especially when you have large amounts of gDNA associate with your sample - on-column DNase dgestions should be used if you really want to get rid of the DNA.