Xiao He:

Senior, I have a question for you: is it accurate to judge the quality of the reverse transcribed cDNA by OD value or electrophoresis?

Xiao Rui:

I have said this many times, so I have to summarize it to avoid repeating it again and again.

Xiao Rui:

1. Running cDNA electrophoresis or measuring the so-called cDNA concentration deeply exposes one's lack of basic knowledge. One needs to learn the course of nucleic acid biochemistry, at least to know the composition of RNA. I have never, and will never, run a cDNA electrophoresis or measure the cDNA concentration to judge the quality of cDNA in my life!

Xiao Rui:

2. The only effective way to judge the quality of cDNA or whether the reverse transcription is successful is the final PCR result. For ordinary PCR results, see who has the brightest amplification; for fluorescent quantitative PCR results, see who has the earliest CT value. If there are other methods, you are welcome to let me broaden my horizons.

Xiaozhi:

When screening the library, it is still necessary to perform electrophoresis on the cDNA

Xiao Rui:

I don't know what a screening library is, but how do you judge the quality of cDNA by running electrophoresis? I want to open my eyes

Xiaozhi:

cDNA electrophoresis is required for third-generation sequencing, size screening, or yeast library construction

Xiao Rui:

Aren't size screening and cDNA quality two different things? You need to screen for size and separate by electrophoresis, which is easy to understand. If you don't run the gel, you can't separate. What I mean is how do you judge the quality of cDNA by running cDNA electrophoresis?

Xiao Rui:

I am against judging whether the reverse transcription is successful or good or bad by running electrophoresis.

Xiao Rui:

It is funny to judge the quality of cDNA by running electrophoresis. Or I want to know what kind of electrophoresis graph represents a good one, and what kind of electrophoresis graph represents a bad reverse transcription?

Xiao Rui:

If we look at the composition of RNA, it becomes clear why the effect of reverse transcription cannot be known by running electrophoresis and measuring OD ratios.

Xiao Rui:

First, the broad definition of total RNA includes 4 types of RNA:

1. Ribosomal RNA (rRNA for short) is what we often call 28S, 18S, and 5S, which accounts for 85% of the total

2. Transfer RNA: tRNA

3. Messenger RNA: (mRNA)

4. microRNA: miRNA

Xiao Rui:

From the total amount, 85% of the total amount is ribosomal RNA, and there are only three sizes, so three bands can be seen, while mRNA only accounts for 1%-5% of the total RNA, and it is the sum of hundreds of expressed genes. Each gene may not even be seen at 0.01%, so no bands can be seen, only tails between 28s and 18s and above and below, smears and background fluorescence.

Xiao Rui:

Then I measured the total RNA for reverse transcription. Only 1-5% of mRNA can be reverse transcribed. 85% are useless 28s, 18s, and 5s. Even if all mRNA is transcribed into cDNA at a ratio of 1:1, you can't see mRNA. Can you see cDNA by doubling it? At most, it will show background fluorescence like mRNA. Is this background fluorescence the same as the background fluorescence of mRNA itself? What's more terrible is that 85% of ribosomal RNA will generally degrade during the transcription process. After degradation, will it form more smears and background fluorescence? How can you tell whether the background fluorescence comes from mRNA, cDNA, or degraded ribosomal RNA?

Xiao Rui:

Moreover, we all know the color enhancement effect of nucleic acid. Degraded RNA, 85% of RNA, will completely cover the existence of cDNA. The color enhancement effect of the absorbance value caused by it will be much greater than the absorbance value derived from cDNA.

Xiao Rui:

For example, if one person scores 10 points and another scores 15 points, the two can still compare and see who is better. But if one person scores 95 points, the 10 and 15 points will immediately become poor students, and there is no difference between them. Just like what Wang Sicong said, when making friends, I don’t care about money. Compared with him, everyone is poor.

Xiao Rui:

This is the reason. If 1% of cDNA is completely covered by 85% of degraded ribosomal RNA, how can you tell whether it is good or bad by running electrophoresis or looking at the OD ratio?

Xiao Rui:

So you can search on Baidu. Can you find the standard for judging the quality of cDNA by running electrophoresis? There is a clear standard for the quality of RNA electrophoresis, the ratio of 28s:18s. Can you find the standard for the quality of cDNA on the Internet?

Xiao Rui:

Different reverse transcription times and different degrees of degradation will result in different electrophoresis of cDNA. The electrophoresis patterns will be very strange. In fact, what you see is not the electrophoresis pattern of cDNA, but the electrophoresis pattern of degraded ribosome RNA. The performance will be different depending on the degree of degradation.

Xiao Rui:

Regardless of whether it is right or wrong, at least it means that many people agree that cDNA cannot be detected by electrophoresis, right? If something is as clear as 1+1=2, there wouldn't be so much controversy.

Xiao Rui:

Xiao Rui:

Xiao Rui:

Someone ran a cDNA electrophoresis diagram. Let's see if it is the same as what I said. Rather than saying that this is a cDNA electrophoresis diagram, it is more like the electrophoresis diagram of ribosomes with different degrees of degradation that I just mentioned.

Xiao Rui:

If you measure the OD of such so-called cDNA, do you think what is measured is cDNA? Or is it the 85% ribosomal RNA that has not been degraded and is still visible?

Xiao Rui:

Xiao Rui:

Xiao Rui:

There are still many people who lack basic knowledge. Diffusion means that the reverse transcription is successful. Degraded RNA is also diffused, right? You just don't add reverse transcriptase, incubate at 42 degrees for 1 hour to see if the RNA will diffuse (degrade). According to these netizens, if it diffuses, the reverse transcription is successful. Then you are surprised to find that it also diffuses without reverse transcriptase. Congratulations, you have obtained cDNA without reverse transcription.

Let’s study hard!

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