Hi, I am doing RNA extraction for my final year project. After extraction of RNA by using conventional method, I did quantification and qualification of RNA by using Nanodrop spec and gel electrophoresis.
Nanodrop did show some RNAs extracted through A260/A280 ratio but there are no bands shown on gel. The agarose gel was 2% and the buffer used was TBE, 100V 30mins for the process.
May I know what will be the possible reasons to cause the scenario? Your replies are much appreciated.
Hi Tiffany
What is the RNA concentration that you obtained?... For gel electrophoresis you still need to load a relative high amount of RNA to see it. Probably you are loading a small amount of RNA
Best.
How many nanogram did you load on your gel?
Did you ladder show up? If yes, then the issue is probably not enough RNA. If no, then you either forgot the Ethidium bromide and/or forgot to turn on the UV light.
How many microlitre did you load in your gel and what is the concentration?
Hi @Francisco J. Enguita @Katie A Burnette @Ghazwan Hasan
Due to my immature skills, the concentration obtained vary from 32.8 ng/ul to 1027 ng/ul and I did believe there is contamination for the extremely high concentration one.
I was loading 5 ul of RNA and 1 ul of GelRed as prestain dye instead of Ethidium Bromide. DNA ladder did show up but not the other samples.
The following attachment was my nanodrop spec and gel electrophoresis result.
Thank you so much for replying me !!
Hi Tiffany.
No problems... it happens. I am doing such things for more than 20 years and sometimes I got these kind of results.
Looking at your gel, there are two lanes that show something. The first lane wish visible sample is clearly degraded (you can see a smear in the bottom of the gel with no defined bands). The second one has a sample that appears to be OK, since you can see bands. I do not know what is the living organism that you used, but it does not look as human to me.
Anyhow... it looks that you have a problem of degradation in some samples.
Regarding the 260/280 relationship, please note that even in degraded samples you can have a good numbers due to the presence of ribose nucleotides. This relationship is just a control for detecting contaminations with other biomolecules.
Hope it helps. Try to repeat the extraction and put lots of care on it.
Best. Paco
The liver is a rich source of nucleases. Your samples seem to be suffering a variable degree of degradation and contamination as well. Tri reagent would be a suitable conventional extraction solution to try. Keep a nuclease-free environment during your extraction.
Francisco J. Enguita
Dear sir,
I am using mice liver and ovaries for my extraction. I would like to ask you one last question regarding the absorbance value of 260 nm and 280 nm.
Is it okay if 260/280 ratio showed around 1.8 -2.0 but absorbance value for 260 nm and 280 nm respectively is more than 1? What does it mean if absorbance value more than 1? Is it because of some kind of contamination?
Thank you, sir ! Good day !
Hi Tiffany
Absorbance at 260 or 280 more than 1 units is a question of concentration. A280 measures proteins (absorbance of tryptophan, phenylanine and tyrosine), meaning that higher values at 280 nm will be related with a protein concentration.
It would be fantastic if you had access to a Bioanalyzer or similar system to really perform a good analysis of the samples, because A260/A280 is just an estimation.
You must have a lot of care in collecting samples, keeping the temperature as low as possible during extraction, specially if you are using non-phenolic extractions.
Good luck
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