thank you so much for the video tutorial! Exactly what I needed as I just started performing RNA extraction and using the Nanodrop! I wanted to learn all the features and how to interpret results!
Thanks for the video, can I quantify mRNA levels from total RNA samples using this method or would I have to isolate mRNA from total RNA then quantify mRNA on it own ?
This depends on your sample and the nucleic acid you are extracting. If you have a high amount of protein in your sample, you need to optimise your extraction method, such as increase the amount of proteinase K and lysis buffer. If you have a high amount of salt then you can reprecipitate the sample and add more ethanol washes. Again this depends on your extraction method. Do you use spin columns?
Very good video!!!! I have a little question that why the result of Nanodrop test is NaN? I couldn't find the answer, I have tested the sample three times and the results are the same, but when I use the machine to test the other sample, the result is pretty normal. I am a little bit confused by this situation
Thanks for your video. I have one question. I get high concentration isolation RNA 114,5 ng/ul. But the purity 260/280 1.90 and 260/230 only 1,4. Can you suggest for increasing purity in 260/230??? What should i have to do?
So a lower than expected 260/230 ratio usually indicates the presence of salts in the sample. How are you extracting your RNA? Usually introducing an additional wash step will improve the ratio
Are you asking about calculating the total amount of RNA in the sample? If so, multiple the concentration (ng/ul) by the total volume of sample (ul) to work out the total amount of RNA in the sample (ng)
hi,, i have one question, i usually extract RNA to do reverse transcript and further real time PCR. the 260/280 values usually i got around 1.9 or 1.8 but the 260/230 sometimes around 1.6 or 1.7. and one of my senior suggest me i can use this one for real time pcr analysis. and i also read in researchgate comments that we can use the 260/230 value around 1.6. what you will suggest me?
Hi Rabia, I would say those values are perfectly fine for RT-PCR. Most people only really look at the 260/280 ratios anyway; yours are spot on. The values I quote in the video are for perfect samples. In the real world these will be slightly different. I would say if the 260/230 ratio was
@@StevenBradburn thank you so much for such a detail reply. by the way i like your videos, its really help me. i hope you will share some more videos on molecular work in future.
The 260/280 ratios are calculated by dividing the measured absorbance at 260 nm by the measured absorbance at 280 nm. DNA and RNA have a slightly different composition, and the different molecular structures absorb light differently. Therefore, they have different ratios.
the fact that Nanodrop doesn't measure factually dna/rna, only ratios, should tell you enough... use qubit, it has kits for dsDNA etc! the reagent binds to actual thing you're measuring. not some random light ratios LOL
This is incorrect. Despite being micro-volume, the NanoDrop is a spectrophotometer and uses the well-known principle of Beer-Lambert's equation and well-defined extinction coefficients to accurately calculate the concentration. The ratios are separate tools, used to evaluate the purity of the sample from common contaminants. The Qubit uses a completely different method, with its own benefits and limitations - the Qubit and NanoDrop are both good instruments, and while they have some overlap in application, the Qubit cannot be used in the same way as a NanoDrop or even do all of the same things (and vice versa).
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thank you so much for the video tutorial! Exactly what I needed as I just started performing RNA extraction and using the Nanodrop! I wanted to learn all the features and how to interpret results!
Very welcome!
Thank you very much! Really useful for mi BSc thesis :)
good and precise demonstration, thankyou
Thanks for this extremely informative video.
Very Well Explained. Thank you so much.
Thanks so much for your explanation.. well understandable❣️❣️
Does this mean that even a well purified RNA sample would contain minute amounts of salts and proteins? BDW thanks for the video. It helped me a lot.
Amazing content!!! Thank you so much!!!!
You are welcome :)
Wow. Awesome. Thanks dude.
أشكرك شكرًا جزيل على الشرح الوافي
Wow. I love this.
Thanks for the video, can I quantify mRNA levels from total RNA samples using this method or would I have to isolate mRNA from total RNA then quantify mRNA on it own ?
What can you say when either of the OD values exceeds the 2.2 mark? Fellow students of mine somehow got samples with a 260/280 value over 6.00
Thank you for your useful information. It is okay if I use your tips to my homework and share with my class?
Yes of course!
Thankyou...very useful 💫💫
You're welcome 😊
Hi! When we found the high concentration of protein or Salt can we clean that sample from protein and salt?
This depends on your sample and the nucleic acid you are extracting. If you have a high amount of protein in your sample, you need to optimise your extraction method, such as increase the amount of proteinase K and lysis buffer. If you have a high amount of salt then you can reprecipitate the sample and add more ethanol washes. Again this depends on your extraction method. Do you use spin columns?
Very good video!!!! I have a little question that why the result of Nanodrop test is NaN? I couldn't find the answer, I have tested the sample three times and the results are the same, but when I use the machine to test the other sample, the result is pretty normal. I am a little bit confused by this situation
nan = zero ng/ul, you have no sample
your sample is blank
Thanks for your video. I have one question. I get high concentration isolation RNA 114,5 ng/ul. But the purity 260/280 1.90 and 260/230 only 1,4. Can you suggest for increasing purity in 260/230??? What should i have to do?
So a lower than expected 260/230 ratio usually indicates the presence of salts in the sample. How are you extracting your RNA? Usually introducing an additional wash step will improve the ratio
Question: how do I calculate the yield of RNA with these results?
Are you asking about calculating the total amount of RNA in the sample? If so, multiple the concentration (ng/ul) by the total volume of sample (ul) to work out the total amount of RNA in the sample (ng)
hi,, i have one question, i usually extract RNA to do reverse transcript and further real time PCR. the 260/280 values usually i got around 1.9 or 1.8 but the 260/230 sometimes around 1.6 or 1.7. and one of my senior suggest me i can use this one for real time pcr analysis. and i also read in researchgate comments that we can use the 260/230 value around 1.6. what you will suggest me?
Hi Rabia,
I would say those values are perfectly fine for RT-PCR. Most people only really look at the 260/280 ratios anyway; yours are spot on.
The values I quote in the video are for perfect samples. In the real world these will be slightly different. I would say if the 260/230 ratio was
@@StevenBradburn thank you so much for such a detail reply. by the way i like your videos, its really help me. i hope you will share some more videos on molecular work in future.
@@rabiazeb9051 you are welcome. And goodluck with your research. That is the plan :) stay tuned for more content
does this apply to DNA as well or it is specific for RNA
Sure. Most information here covers DNA too. Main difference is that pure DNA has a 260/280 ratio of 1.8, whereas pure RNA has a 260/280 ratio of 2
Why is RNA’s ratio of 260/280 higher than DNA what makes one higher than the other?
The 260/280 ratios are calculated by dividing the measured absorbance at 260 nm by the measured absorbance at 280 nm. DNA and RNA have a slightly different composition, and the different molecular structures absorb light differently. Therefore, they have different ratios.
the fact that Nanodrop doesn't measure factually dna/rna, only ratios, should tell you enough... use qubit, it has kits for dsDNA etc! the reagent binds to actual thing you're measuring. not some random light ratios LOL
This is incorrect. Despite being micro-volume, the NanoDrop is a spectrophotometer and uses the well-known principle of Beer-Lambert's equation and well-defined extinction coefficients to accurately calculate the concentration. The ratios are separate tools, used to evaluate the purity of the sample from common contaminants. The Qubit uses a completely different method, with its own benefits and limitations - the Qubit and NanoDrop are both good instruments, and while they have some overlap in application, the Qubit cannot be used in the same way as a NanoDrop or even do all of the same things (and vice versa).