How To Interpret Nanodrop Results For RNA

This video tutorial is part of the Mastering qPCR Online Course in The Top Tip Bio Academy.
🎓 MASTERING QPCR ONLINE COURSE 🎓
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In this video, I will explain what the Nanodrop results mean and how you can use them to deduce what is actually in your sample.
I will explain what the 260/280 and 260/230 ratios are and what you should look out for in good RNA samples.
I will also give you 8 tips when using the Nanodrop to improve your results.
THE NANODROP RESULTS EXPLAINED
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8 TIPS FOR USING THE NANODROP TO MEASURE DNA/RNA
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MORE HELPFUL HINTS & TIPS
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[@StevenBradburn]

🎓 MASTERING QPCR ONLINE COURSE 🎓 courses.toptipbio.com/p/mastering-qpcr

[@keshikesh8667]

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.

[@lauradelasheras1492]

Thank you very much! Really useful for mi BSc thesis :)

[@89lilly]

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!

[@StevenBradburn]

Very welcome!

[@lilianmusembei8490]

good and precise demonstration, thankyou

[@jensatzema2625]

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

[@zuleykhasalim6196]

Thanks so much for your explanation.. well understandable❣️❣️

[@lewp4452]

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 ?

[@marwaabdelhamied6607]

Thanks for this extremely informative video.

[@ajmalkhan5651]

Very Well Explained. Thank you so much.

[@reviremin3672]

أشكرك شكرًا جزيل على الشرح الوافي

[@angelosgavrielatos4343]

Amazing content!!! Thank you so much!!!!

[@StevenBradburn]

You are welcome :)

[@HaroonKhan-cy1mi]

Hi! When we found the high concentration of protein or Salt can we clean that sample from protein and salt?

[@StevenBradburn]

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?

[@tranlamvy6498]

Thank you for your useful information. It is okay if I use your tips to my homework and share with my class?

[@StevenBradburn]

Yes of course!

[@cnsisow]

Wow. I love this.

[@nom3nnescio]

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

[@Merrexz]

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).

[@Nicole99231]

Why is RNA’s ratio of 260/280 higher than DNA what makes one higher than the other?

[@Merrexz]

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.

[@NurulHikmawati]

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?

[@StevenBradburn]

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

[@osmirod1]

Wow. Awesome. Thanks dude.

[@HimeBaezChan]

Question: how do I calculate the yield of RNA with these results?

[@StevenBradburn]

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)

[@yimingyin4080]

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

[@nom3nnescio]

nan = zero ng/ul, you have no sample

[@nom3nnescio]

your sample is blank

[@rabiazeb9051]

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?

[@StevenBradburn]

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

[@rabiazeb9051]

@@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.

[@StevenBradburn]

@@rabiazeb9051 you are welcome. And goodluck with your research. That is the plan :) stay tuned for more content

[@xavierrosas2825]

does this apply to DNA as well or it is specific for RNA

[@StevenBradburn]

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

[@anitasakha9674]

Thankyou...very useful 💫💫

[@StevenBradburn]

You're welcome 😊