The low ratio of A260/A230 is usually caused by impurities with the maximum absorption wavelength at 230nm. Let’s see what these impurities include:

• Common pollutants	Absorption wavelength	Ratio effect
  Protein	~230nm and 280nm	Simultaneous reduction of A 260 /A 280 and A 260 /A 280 ratios
  Guanidine salt	220-240 nm	Reduce the A 260 /A 280 ratio
  Phenol	~270nm	-
  Trizol	~230nm and 270nm	Reduce the A 260 /A 280 ratio
  EDTA	~230nm	Reduce the A 260 /A 280 ratio
  Ethanol	230-240 nm	Reduce the A 260 /A 280 ratio

 
 
 
Absorption wavelength and contrast value of common pollutants

Protein contamination
Protein pollution can be regarded as the most common pollution in the nucleic acid extraction process. Protein exists between the upper aqueous phase and the lower organic phase . Pollution will reduce the ratio of A260/A280 and A260/A230 at the same time, and the ratio of A260/A230 will change more obviously than the ratio of A260/A280.
During subsequent reverse transcription or qPCR reactions , protein residues can inhibit or interfere with enzyme function . The best way to avoid protein contamination is to keep in mind the principle of “rather less than more, a small amount many times” when aspirating the supernatant.

2. Guanidinium pollution
hydrochloride (GuHCl) and guanidine thiocyanate (GTC) have the effect of denaturing proteins, which can quickly destroy cell membranes during the nucleic acid extraction process, causing protein denaturation and precipitation. The absorption wavelength of GuHCl and GTC is between 220-240 nm, and the residual guanidinium salt will reduce the ratio of A260/A230 . Although the residual guanidinium salt will reduce the ratio, the impact on downstream experiments is actually negligible .

3. Trizol contamination
The main component of Trizol is phenol . The main function of phenol is to lyse cells and release proteins and nucleic acid substances in cells. Although phenol can effectively denature proteins, it cannot completely inhibit RNase activity. Therefore, 8 -hydroxyquinoline , guanidine isothiocyanate , β- mercaptoethanol, etc. are added to TRIzol to inhibit endogenous and exogenous RNase.
When extracting cellular RNA, Trizol can rapidly lyse the cells and inhibit the nuclease released from the cells, and the residual Trizol will significantly reduce the ratio of A260/A230.
Processing method: When centrifuging, it must be noted that the phenol in Trizol is easily soluble in the water phase under the condition of 4° and room temperature.

4. Ethanol residue
Ethanol is used in the final process to precipitate the DNA while dissolving salt ions that may be bound to the DNA. The absorption wavelength of the highest absorption peak of ethanol is also at 230-240 nm, which will also reduce the ratio of A260/A230 .
The method of avoiding ethanol residue can be repeated twice during the final elution, blowing in the fume hood for two minutes to allow the ethanol to fully evaporate before adding buffer for elution.
However, it should be known that the ratio is only an evaluation index of RNA quality. If the above-mentioned operations are strictly regulated, the deviation between the ratio and the standard range will not have a great impact on downstream experiments.
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