I've been working with RNA agarose gel electrophoresis and having trouble to visualize positive RNA control samples(1ug) and RNA ladder(3ug). enclosed is the best gel image I've got (please ignore lane 3-5). The gel is stained in Gelstar(10X sensitivity to EtBr) in this case. I also tried EtBr post-electrophoresis staining and got even worse result. either case, the positive RNA and RNA ladder stained very poorly. and Ladder lanes are indiscernible. below is some other info how I ran the gel:
1% agarose gel, with formaldehyde and reduction buffer from Northernmax
RNA ladder (3ug from invitrogen), RNA positive control (1ug) from Northernmax kit
3X volume of formaldehyde loading dye
65C water bath 15min prior to sample loading
5V/ cm electrophoresis
MOPS 1X buffer in DEPC H2O
Major question:
1\ what's the reason that 1ug/ug RNA control/ladder wont give clear bands?
Other questions:
2\ the 1ug RNA control in lane 1, it seems intact and not smeared. does that indication no degradation?
3\ I used the agarose provided in the northernmax kit to run RNA gel. Can I use regular agarose (used for DNA) to run RNA gels?
The major problem with visualizing RNA in an agarose gel is that the dye (Gelstar, EtBr, etc..) doesn't intercalate as well into RNA since it is single stranded compared to DNA which is double stranded. This is especially bad when you stain the gel post-running.
If you are just checking the integrity of your RNA, you can add your dye (Gelstar, EtBr, etc..) to your gel prior to running your samples. 1ug of total RNA should be easily visible under UV.
If you are running an agarose to blot, you can add EtBr to your sample after you heat at 65C and then load your sample into the gel (no dye added). The RNA will bind the EtBr and be visible under UV light and you will not have to stain post-running. I'm not sure if you can do it this way with Gelstar.
You can use the most common types of agarose (except low melting point) for running RNA. The RNA you have in the first two lanes looks to be intact with no degradation.
Good Luck!
Hi, If you only need to check our RNA integrity I suggests you to use a common agarose gel.
In my experience I normally prepare 1%agarose with EtBr, with fresh TBE buffer, in a cleaned (it is really important when you run RNA) electrophoresis apparatus. I didn't use a ladder or dye but I only add milliQ water and autoclaved glicerol to my RNA and load in the gel. In a small gel (as the one in your figure) I run for 30 min at 50V, without dye I known that, with this condition, I have a good separation of the RNA bands. As Tom said you have to see very easily your RNA, in particular 3ug!
There are several threads on ResearchGate with similar questions. (Use the dropdown menu in the Search box to select "questions" and then type in your search key words.)
The first of these two links is particularly useful:
https://www.researchgate.net/post/Can_anyone_help_me_with_gel_electrophoresis_of_total_RNA
https://www.researchgate.net/post/How_can_I_check_the_RNA_integrity_and_know_if_the_observed_bands_are_from_DNA_contamination_or_not/1
What is your UV light source? You need a short wave (254 nm) or medium wave (~300nm?) UV light to see EtBr fluorescence. The color of your bands looks unusual--should be bright orange, so it's also possible that the color filter you are using for photography is not correct.
@David f Barker, thanks for the great point about using the right wavelength to visualize. the gel in the image is stained with GelStar which relies on blue light instead of UV. the blue light is filtered by an amber filter that's why the background is a bit orange. I've tried EtBr post-staining with UV, result even worse.
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