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About Nucleic acid extraction

The beginning and end of purified nucleic acid

Everything is difficult at the beginning, purified nucleic acid is the most

Initiation of molecular experiments, for subsequent experiments

Success or failure has a crucial impact. 

Trace/ultra-trace UV spectrophotometer is favored by many scientific researchers because it can detect nucleic acid concentration and protein and salt ion residues simply, quickly and economically. 

As we all know, A260 means nucleic acid absorbance OD value, A280 means protein concentration absorbance OD value, A230 means salt ion concentration absorbance OD value, so A260 can convert nucleic acid concentration, A260/280 means protein residue, A260/230 means salt ion residue, but this is just a rule discovered by researchers based on a large number of measurement results, and it is ” conventional ” to believe that it can explain the purity of DNA and RNA to a certain extent . It is not the only standard for the identification of nucleic acid yield and purity, it is only used as a reference, and it is not a “gold standard”.

derthfd (1)

The Thermo Fisher ND-2000 manual emphasizes the reliability of the test results 

The reason is that the results obtained by using a micro/ultramicro UV spectrophotometer only reflect the yield and purity of nucleic acid from the side, and there are many influencing factors (optical path, sample volume, sample concentration, pH value, other ions that affect absorbance measurement, etc.) , the measured OD value is not necessarily accurate. At the same time, due to the lack of specificity of the absorbance value determination material, single-stranded DNA, double-stranded DNA, RNA, dNTPs, and some proteins all have absorption peaks at 260nm wavelength, and the concentration determined by A260 alone is not necessarily the real DNA or RNA. Concentration, and its integrity and degradation cannot be judged.

So how to judge the quality of our purified nucleic acid? In addition to using a micro/ultramicro UV spectrophotometer for detection, methods such as agarose gel electrophoresis are also required to visually display the purity and integrity of the nucleic acid, and to indicate the concentration to a certain extent. In this way, the nucleic acid yield and purity obtained from the detection results of various methods are credible.

Experimental chart comparison

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Genomic DNA of H1299 cells was extracted using company A’s DNA Extraction kits, and significant impurity contamination was seen, resulting in high OD values

(OD measurement value and corresponding electropherogram)

Evaluation index

Is there a “gold standard” for nucleic acid yield and quality testing?

As far as we know, there is no simple, fast and cheap method. Whether it is spectrophotometer, gel electrophoresis, or mass spectrometry, they all reflect the concentration and purity of nucleic acid in the solution from different aspects, and they need to be judged by referring to each other.

The best evaluation index is always the follow-up experimental application . It does not affect the efficiency of reverse transcription and PCR amplification. Obtaining reliable amplification products and Ct values, and obtaining a complete sequence by sequencing is successful nucleic acid extraction.

To sum up, the view of ratio-only theory should be challenged by the view of “using good downstream experimental results as good extraction parameters”! 

Recommended Foregene DNA RNA extraction kits to get high DNA/RNA purity:

https://www.foreivd.com/reagent/dna-isolation-series/

https://www.foreivd.com/reagent/rna-isolation-series/


Post time: Dec-14-2022