260/230 values of RNA and precipitation of RNA - (Jun/05/2015 )

Hello again,

I have extracted RNA and i am satisfied with the purity (260/280 ratio) and the yield. What is troubling me is the very low 260/230 ratio? Therefore, i wanted to know how important is the 260/230 ratio in RNA extraction. I will use the RNA for qPCR.

Yield 26-35 ng/ul and 260/280 ratio is between 1.9-2.2

If i want to re-precipitate the RNA should i use sodium acetate or Lithium chloride to do this?

Thanks

It is usually assumed that the 230 absorbance is carbohydrates. Poor 260/230 may not be a problem for qPCR. To test this, try qPCR with a dilution series of your RNA (1:1, 1:2, 1:10, 1:100). If Ct values scale with the dilutions and the curves have the same shape, whatever is absorbing in the 230 isn't causing a problem.

If more purification is needed, Lithium chloride precipitation may help to selectively precipitate RNA from carbohydrates. Normal sodium acetate precipitation is not likely to work. Instead, high potassium acetate (0.8-1.5M) with about 1.4M NaCl will probably have better results.

The higher 230 value can also be due to salts. Did your RNA purification procedure use isopropanol instead of ethanol? If so, that's probably your issue. Isopropanol increases yield of nucleotides, but also salts, so purifications with isopropanol usually have a bad 260/230 ratio (usually <1 in my experience). If all your samples have similar 260/230 ratios and concentrations, and you're comparing between samples, then your qPCR data will probably be okay. A dilution series is a good control.