My A260/A230 ratios are within the range of 2 to 2.2 for my RNA extraction from isolated CD4 cells. However my A260/A230 are in decimals, far below 2. Must I get the range of both ratios right? Which ratio is more important?
Hi Martin,
I usually go with 260/280. This ratios is for the four nucleotides present. The accepted ratio range can vary depending on your next experiment but 1.8-2.0 is considered good. RNA will typically have a higher ratio due to Uracil compared to that of Thymine.
260/230 - It's a secondary measure to confirm the purity. The 260/230 ratio are usually higher than 260/280 ratio. Expected range for this ratio is 2.0-2.2. If your ratio is significantly lower as you mentioned, its an indication that there may be some contaminants that absorb at 230 nm.
Among the most common contaminants are EDTA, carbohydrates and phenol all have absorbance near 230 nm.
Hope this helps.
Both the A260/A280 and A260/A230 ratios are important in assessing the quality of RNA or DNA extraction.
The A260/A280 ratio is used to assess the purity of the RNA or DNA sample. The ratio is calculated by dividing the absorbance at 260 nm (A260), which indicates the presence of nucleic acids, by the absorbance at 280 nm (A280), which indicates the presence of protein contaminants. A ratio of 1.8-2.0 is optimal for RNA, while a ratio of 1.8-2.2 is optimal for DNA. A lower ratio indicates the presence of protein or other contaminants that may interfere with downstream applications.
The A260/A230 ratio is used to assess the presence of contaminants such as salts, residual phenol, or other organic compounds. The ratio is calculated by dividing the absorbance at 260 nm (A260) by the absorbance at 230 nm (A230). A ratio of 2.0-2.2 is considered optimal for RNA, while a ratio of 1.8-2.0 is considered optimal for DNA. A lower ratio may indicate the presence of contaminants that can interfere with downstream applications.
Therefore, both A260/A280 and A260/A230 ratios are important in assessing the quality of RNA or DNA extraction, as they provide complementary information about the purity and presence of contaminants in the sample.
Hello Martin Akandawen
Whenever we refer to RNA quality for any RNA-based application, we are referring to both RNA integrity as well as RNA purity.
Even when the integrity of RNA is good, the RNA can suffer from a number of impurities, including organic solvents and salts left over from extraction (e.g., phenol, chloroform), genomic DNA, nucleases as well as other proteases that can make it through the extraction process. All of these impurities pose potential problems with downstream processing steps, for instance, by inhibiting or digesting the enzymes that carry out reverse transcription and library synthesis, or by skewing the data, for example, sequencing reads originating from contaminating genomic DNA.
For 260/280 nm ratio, a ratio of 2 is usually interpreted as pure for RNA. Lower ratios may indicate the presence of proteins or other contaminants that absorb strongly at or near 280 nm.
For 260/230 nm ratio, a ratio of 2-2.2 is generally accepted as pure RNA. Lower ratios indicate the presence of contaminants such as EDTA, carbohydrates and phenol, TRIzol and others that absorb near 230 nm.
So, you will have to get the range of both ratios right. Both are important for RNA-based application.
Best.
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