Hi everybody,
I would like to ask you, if you have some experiences in dsRNA quantification. We´re using nanodrop normally, however i´m really interested, if I choose in settings "RNA", would it be the correct concentration - because it´s meant to be ssRNA, isn´t it? How´s your experiences? Is it necessary to calculate it in a different way?
Thanks for your advice.
Sincerly,
Matej Medla
As is the case for DNA, the ds form has a lower extinction coefficient than the ss form. Thus, you shouldn't use the pre-pregrammed RNA setting of the NanoDrop. Instead, record the actual absorbance value and mutiply it by the conversion factor adequate for ds RNA. Nwokeoji et al. 2017. Anal Chem. 89, 13567-13574 determined a factor 46.18−47.29 μg/mL/A260 for ds RNA, which is close to the value of 50 μg/mL/A260 generally accepted for ds DNA.
RNA is usually single-stranded, although there may actually be double-stranded forms due to hybridization or higher structure. If your question about dsRNA quantification refers to mass weight (ng or ug), but not molecular molar quantity, I think both double-stranded and single-stranded RNA are the same by using nanodrop to measure. For example, you mix 1 ug of sense ssRNA (a single specific kind of RNA) with 1 ug of the ss-antisense of that RNA, and they are 100% paired to form a dsRNA, you will get 2 ug of RNA. Although it has not changed molecularly in quantity, its mass will still increase.
Thanks for references, Pierre Béguin I have already read similar conversion as you mentioned here and i was curious if it´s really necessary - however, you think it´s maybe better to set "DNA" for dsRNA quantification? Kai Li thanks a lot - you made me calm :D i usually calculate with the ammount which i got in RNA messurement. But i will deffinitelly confirm it soon, thank you both!
Regarding your question, if you are really fussy about the quantification, you can tale the absorbance value given the Nanodrop and use the coefficient of Nwokeoji et al. However, given the likely error of the Nanodrop value, for most cases a systematic error of 5-8% will probably not affect your result. Thus, if you prefer to use one of the pre-set calculations of the Nanodrop, you should choose the dsDNA setting.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA