Why I could not observe the tRNA in the well of gel agarose electrophoresis?

17 December 2022 8 7K Report

I did several times gel agarose electrophoresis to find the best N/P ratio of chitosan/tRNA polyplex. I don't know why I could not observe the tRNA in the well of gel agarose electrophoresis and the whole gel matrix? Even there is no smearing.

Would you please help me to interpret my result?

Thanks for your time and consideration.

Zahra Kariminezhad

I've made a correction in the question. The sequence which I use in this gel is tRNA and 72 nucleotides. The agarose gel is 4%. I saw this problem in the mRNA and siRNA polyplex too.

I don't know why the tRNA in the polyplex is not visible in the well and the gel matrix.

Laura Leighton

Can you explain exactly what is on this gel? It would be a lot easier to intepret this if I knew: what's the ladder, what's in the other wells that have something in them, and what was the gel stained with? Was the stain added to the liquid gel before pouring or did you soak the gel in a buffer containing stain? Also, since you mention NP ratio - was anything loaded on this gel actually complexed with some kind of delivery reagent/nanoparticle/etc when it went into the wells?

Zahra Kariminezhad

Dear Laura, Thank you for your help.

Regarding you questions, I have written the answers as follows:

1- Gel is 4% agarose gel

2- 1st Lane is Low DNA Ladder, 2nd lane is free tRNA, 3rd lane up to lane 8th are chitosan/tRNA polyplex with N/P 1, 2, 3, 4, 5, and 10.

3- All polyplex samples (N/P 2-10) were prepared and after incubation for 15 minutes and mixing with loading buffer injected right away to the wells.

4- The gel was stained with a solution of ethidium bromide.

5- The ladder is Invitrogen™ Low DNA Mass Ladder

As seen in the image, almost all tRNA are complexed with chitosan at N/P ratio 2 (red rectangle) but I could not see the tRNA polyplex at the well too. But other polyplex samples (N/P 3-10) are visible clearly at the wells. I did the electrophoresis several times and I have the same problem.

Laura Sánchez-Hechavarría

You might consider to use a bigger concentration of agarose, unless that is what your experiment required. What I see is that the substance you are using to stain the gel is not working, so maybe it is been inhibited by your treatments. So you might use another for this purpose.

Laura Leighton

Hmmm, interesting. It's odd that it's just one lane, while the results for all the others make sense (large complexes being trapped in wells is pretty typical.) Have you seen this result multiple times? Are you sure it is not a random sample or loading failure? You could try a gel stain that is more sensitive and better at detecting single stranded nucleic acids (SYBR gold would be my choice here).

Zahra Kariminezhad

Thank you, Laura Leighton and Laura Sánchez-Hechavarría .

Actually, I do not have other stain like SYBR gold to do. But I will try to order that and see the difference. I did this test several times with almost same results. For me, it is confusing too.

Anyway, I will repeat this test again and will share with you my results.

Nikolay Klyashtornyy

There could be several reasons why you are unable to observe tRNA in the well of agarose gel electrophoresis:

Loading volume: Ensure that you have loaded a sufficient volume of the tRNA sample into the well. If the volume is too low, the concentration of tRNA might be below the detection limit, making it difficult to visualize. Try increasing the volume of your sample loaded into the well.

Concentration of tRNA: Check the concentration of your tRNA sample. If it is too low, it may not produce a visible band on the gel. Consider concentrating the tRNA sample or optimizing the extraction/purification method to increase its concentration.

DNA stain compatibility: Confirm that the DNA stain you are using is compatible with tRNA detection. Different stains may have varying affinities for different nucleic acids. Ensure that the stain you are using is suitable for visualizing tRNA.

Gel concentration and running conditions: Evaluate the concentration of agarose in your gel. If the concentration is too high, it may impede the migration of smaller-sized molecules, such as tRNA. Consider using a lower percentage agarose gel to improve resolution. Additionally, check the running conditions, such as buffer composition, voltage, and run time. Adjusting these parameters might improve the migration and visualization of tRNA.

Sample preparation and loading: Ensure that your tRNA sample is properly prepared for electrophoresis. It should be denatured, either through heat or chemical denaturation, to ensure proper migration during electrophoresis. Confirm that you are loading the sample directly into the well without any leakage or loss during the loading process.

Detection method: Consider alternative methods for visualizing tRNA. If standard DNA stains are not yielding results, you may explore other detection methods specific to tRNA, such as northern blotting or hybridization with labeled probes.

It is worth noting that tRNA is relatively small in size, and depending on the experimental conditions, it may migrate differently compared to larger DNA fragments. Ensure that your gel electrophoresis system and parameters are suitable for the size range of tRNA.

Overall, it is important to troubleshoot and optimize various factors including sample concentration, staining method, gel conditions, and loading techniques to enhance the visualization of tRNA on the agarose gel.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

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