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Recently, I discovered something amazing! Many advanced experiment professionals around him don’t even know some very basic experimental knowledge points.

For example, can you answer the following questions?

Is there a difference between OD260 and A260? What does each mean?
OD is the abbreviation of optical density (optical density), A is the abbreviation of absorbance (absorbance), the two concepts are actually the same, “optical density” is “absorbance”, but “optical density” is in line with most national standards and more standardized.

Usually we measure the OD value at 260nm to calculate the nucleic acid concentration, so what does 1OD represent?
Nucleic acid has a maximum absorption peak at a wavelength of 260nm, which contains both DNA and RNA, as well as fragmented nucleic acid fragments (this is the key point).
The OD value measured at a wavelength of 260 nm was recorded as OD260. If the sample is pure, the OD260 value can calculate the concentration of the nucleic acid sample.
1 OD260=50 μg/ml dsDNA (double-stranded DNA)
=37 μg/ml ssDNA (single-stranded DNA)
=40 μg/ml RNA
=30 μg/ml dNTPs (oligonucleotides)
Is there any connection and difference between RT-PCR, Realtime-PCR and QPCR?
RT-PCR is short for Reverse Transcription PCR
Real Time PCR=qPCR, short for Quantitative Real Time PCR
Although Real Time PCR (real-time fluorescent quantitative PCR) and Reverse Transcription PCR (reverse transcription PCR) both seem to be abbreviated as RT-PCR. But the international convention is: RT-PCR specifically refers to reverse transcription PCR.

What are the commonly used nt, bp, and kb to describe the length of DNA/RNA in biology?
nt = nucleotide
bp = base pair base pair
kb = kilobase

Of course, you would say that many people don’t care about these small details! Everyone does this, and no one will ask you what it is. You know this is unnecessary, right?

No, No, No, it is very necessary to know this! because of which?
Because you want to post an article! Brother! Whether you are aiming for graduation or pursuing scientific research achievements, you must rely on articles to speak!

Nucleic acid extraction should be the simplest and most basic experiment. The quality of nucleic acid extraction directly determines the results of subsequent experiments.

Although I have said it many times, there are still many friends who don’t care. This time I decided to move out of the article!

image1
Minimum Information for Publication of Quantitative Real-Time PCR Experiments, referred to as MIQE, is a set of fluorescence quantitative experiment guidelines launched internationally, which proposes minimum standards for the experimental information necessary for evaluating fluorescence quantitative PCR experiments and publishing articles. Through the experimental conditions and analysis methods provided by the experimenter, the reviewers can better evaluate the validity of the researcher’s experimental scheme.
image2
It can be seen that in the section of nucleic acid extraction, the following detection items have been proposed,

“E” indicates information that must be provided and “D” indicates information that should be provided if necessary.

The form is very complicated, in fact, I want to say that everyone needs to start from

purity (D),yield (D), integrity (E) and consistency (E) to evaluate Nucleic acids in these four aspects.

According to experimental habits, first talk about the evaluation methods of purity and concentration.

OD measurement is the favorite and easiest detection method for experimenters. As for the principle, I won’t go into details here. Many laboratories now use ultra-micro spectrophotometers to directly quantitatively analyze nucleic acid samples. While displaying the absorbance value, the program directly gives the concentration value (nucleic acid, protein and fluorescent dye) and related ratios. As for the analysis of the OD value, Save this picture and you’ll be fine.

Universal OD value solution list

image3However, there are a few caveats that need to be brought out separately for you.

(After all, I know you must be the ones who save up and wait until you need them!)

Note 1 Equipment

  The OD value will be affected by different equipment. As long as the OD260 is within a certain range, the values of OD230 and OD280 are meaningful. For example, the absorbance range of the common Eppendorf D30 at 260nm is 0~3A, and the NanoDrop One of Thermo is at 260nm. The absorbance range of 0.5~62.5A.

Note 2 Dilution reagent

The OD value can be affected by the dilution of different reagents. For example, the OD260/280 reading of purified RNA in pH 7.5 10mM Tris buffer is between 1.9-2.1, while in neutral aqueous solution the ratio will be lower, maybe only 1.8-2.0, but this does not mean that the quality of RNA changes Difference.

Note 3 Residual substances

The existence of residual substances will affect the accuracy of nucleic acid concentration measurement, so it is necessary to avoid protein, phenol, polysaccharide and polyphenol residues in nucleic acid samples as much as possible.

However, in fact, extraction with organic reagents is an old method. In commercial kits, the extraction effect can be achieved through a silica-based adsorption column combined with centrifugation, avoiding toxic and harmful organic reagents that are difficult to remove, etc. The problem, such as Foregene’s nucleic acid extraction kit, does not use DNase/RNase and toxic organic reagents throughout the operation, fast and safe, and the effect is good (accidentally said that it was bald, but I know you want to know).

Example 1: Genomic DNA extraction yield and purity

Foregene Soil DNA Isolation Kit (DE-05511) treats soil samples from various sources, and the amount and purity of genomic DNA obtained are shown in the following table:
image4Example 2: Tissue RNA extraction yield and purity

Animal Total RNA Isolation Kit (RE-03012) processed various tissue samples, and the amount and purity of RNA obtained are shown in the table below (for mouse tissue):
image5However, don’t think that you are done with the OD value. Do you have any take care of the key points I drew for you in the front?

Notice

Fragmented nucleic acid molecules will also be calculated in the absorbance. Assuming that you have genomic DNA residues in the RNA, your OD value will appear to be very high, but the actual concentration of RNA cannot be determined. Whether your RNA is It is not clear whether there is degradation, so we still need a comprehensive evaluation method to give a more accurate judgment, that is, the nucleic acid integrity evaluation mentioned in MIQE.


Post time: Jan-13-2022