EtOH precipitation for RNA purification

How to check the purity of nucleic acid?

Once you check 260/280 and 260/230 ratios with NanoDrop, you will see the purity of your DNA or RNA sample.

A 260/280 ratio of ~2.0 is generally accepted as “pure” for RNA.

The 260/230 value are commonly in the range of 2.0-2.2.

If the ratio is a lot lower than 2.0, you can process RNA purification.

You can see the purity of your samples with NanoDrop. However, it only shows the ratios and concentration. You can't check its integrity with NanoDrop.

Before Start

Please note that EtOH precipitation only occurs when a minimum concentration ~10 ug/ml of RNA is present as this is concentration dependent. If the concentration of the sample was too low, add glycogen. Also, if you have too low amount of RNA, you will lose most of the RNA during the precipitation.

Materials

100% EtOH or isopropanol

70% ice cold EtOH

Nuclease free DW

NaOAc (pH 5.5) or other salts (see the table below)

(Optional) glycogen

Procedure

1. Add 0.1 volume of 3 M sodium acetate. Note1. If the RNA concentration is too low, add glycogen to a final concentration of minimum 20 ug/ml. Glycogen does not interfere with spectrophotometric readings, electrophoresis, and most molecular biology enzymatic applications including PCR. Note2. Salts neutralize the charges on the sugar-phosphate backbone of nucleic acid. A commonly used salt is sodium acetate. In solution, sodium acetate breaks up into Na+ and [CH3COO]-. The positively charged sodium ions neutralize the negative charge on the PO4- groups on the nucleic acid, leading the molecule far less hydrophilic.

2. Add 2.5 volume of 100% EtOH. Note1. 1 volume of isopropanol can be used instead of ethanol. Note2. EtOH makes it much easier for Na+ to interact with PO4. This makes the nucleic acid less hydrophilic, thus causing it to drop out of the solution.

3. Brief vortex for 2 sec and incubate at -20℃ for over 1 hour. Note. You can stop at this step for several days. For long term storage, keep at -80℃.

4. Centrifuge at 12,000g for 30 min at 4℃.

5. Remove sup and add 1 ml of ice-cold 70% EtOH.

6. Brief vortex for 2 sec.

7. Centrifuge at 12,000g for 5 min at 4℃.

8. Remove sup and brief spin down. Note. There are lots of invisible residue RNA stuck on the wall. To get the RNA as much as possible, brief spin down after removal of 70% EtOH. This step will dramatically increase your yield.

9. Remove residual ethanol.

10. Add nuclease-free water without air-drying. Note. As I mention in earlier post, I don't think we can fully remove residual EtOH with 5 min air-dry but also I think RNA pellet becomes too dried to be properly dissolved in DW. Thus, I added remove residual EtOH after additional spin down without air-drying.

The 260/280 ratio is ~2.0 and 260/230 ratio is less than 1.6 (figure 1). However, the ratios are increase after EtOH precipitation for RNA purification (figure 2).