Cannot isolate total RNA using column - (May/22/2013 )

Hi all,

I am trying to isolate total RNA from Staphylococcus strain, but every time I use the spin column, the yield is extremely low, closed to nothing. Does anyone know why?

I tried no-column preparation, then I saw big peaks with A260/A280 > 1.8. However, I need to make sure there's no DNA contamination as I need to do RNA-seq afterwards. How can I digest the DNA without the involvement of column?

Thank you very much in advance for your help!

Best,
Littlepumpkin123

For a standard (acidic phenol:chloroform extraction, commonly known as trizol and similar reagents) the differentiation between DNA and RNA is due to the pH of the buffered phenol used in the extraction. The RNA should be retained in the aqueous phase and the DNA should be in the white interface during the phase separation step. The best way to ensure that you do not have DNA carry-over is to make sure that you do not pick up ANY of the white interface. It should be possible to add a DNase step somewhere, but DNases are often relatively non-specific and will degrade RNA as well.

The A260/A280 thing is a bit problematic - it depends on the pH of the solution you are measuring the absorbance in, if you use water you will get a different reading to using TE or other similar reagents due to a shift in the absorbance of the A280 peak for potential contaminants. For pure RNA in correctly made TE the ratio should be 2.0. A good explanation can be found here.

Thank you Bob1!

I was using water to dissolve the final product. I think I'll try not using DNAse and use PCR to verify the results then.