I ran my total RNA sample in a denaturing RNA gel (MOPS/formaldehyde gel) and I came across two problems:
(1) It was run at 60 V (10 cm gel). The samples are very slow to migrate, ~3 hrs to run into the middle of the gel;
(2) The background EtBr fluorescence is very strong (bright yellow to orange) compared with the regular DNA agarose gel. Since the migration is so slow, EtBr only moves 40% when the bands move 50% from the tops of the gel.
Does anyone have similar problems when running a RNA gel in MOPS? Could you suggest any ways to improve it?
Hello Garima, Thank you for your wonderful help. I ran 1% agarose gel. I will try stain after running. I just ran my RNA sample on DNA gel today. It still runs as slow as the denaturing gel. I guess it is due to formamide in the sample. I saw two very bright bands, but there some smear start from the well till the lower band. I'm not sure if the degradation happened during the run ( in the gel), or happened in the sample itself.
I suggest you to prepare 1.2% formaldehyde denaturing agarose gel.Gel electrophoresis can be carried out in a formaldehyde running buffer at 45 volt for an hour. Then, agarose gel need to be destained for 30 min in an autoclaved DEPC-treated water and view using gel documentation system. Good luck.
http://everest.bic.nus.edu.sg/hons99/gekming/protocol/node8.html
Formaldehyde will hydrogen bond with the bases and denature RNA, giving single-stranded RNA. RNA is usually run on a denaturing gel because it forms extensive secondary structures which significantly affects the rate of migration on a gel, unlike DNA which is predominantly linear.
Heating denatures the RNA and allows the EtBr to intercalate and run with the RNA
Try loading the RNA sample in 1% agarose gel. Use TBE as the tank buffer. Run the gel for 200V for 20 min. You can either do a post-staining with EtBr or add the EtBr in the gel before running.
Hello Wang, use 1.2% agarose-formaldehyde gel. Run the gel for 120 V and can go for post staining or EtBr in the gel.
You can either incubate gel after run in water to get rid off the background (usually 2x15 min on slow shaking helps) or stain the gel after run in EtBr solution. Then you will see probably some background that you can eliminate by shaking it in water. What was agarose gel %, you should run 0.5-0.8. More than that for total RNA will not give good result
Thank you so much for all your great suggestions! I will try it again and let you know how it looks!
Hi Suning,
what you actually need is denaturing of RNA. There are two other, than formaldehyde, ways. Just heat the RNA sample in 0,2 mM EDTA (pH 7) at 70*C for 3 min, put samples into ice water, then load and run samples on 1% agarose gel. You can use Tris-borate or sodium acetate buffer system for electrophoresis. Best way is denaturing of RNA in presence of glyoxal in presence of DMSO: mix 2.5 mmL of RNA in RNase free water (0.5-1mmG of RNA) with 5.5 mmL DMSO + 1.9 mmL 40% deionized glyoxal + 1.1 mmL 100 mM sodium phosphate buffer (pH 7). Incubate sample at 56*C for 50 min, chill to 20*C + 2.5 mmL solution of 2.5% bromphenol blue-40% sucrose-50 mM sodium phosphate (pH 7). Then run electrophoresis. You can run gel in presence of ethidium bromide and washing out access of fluorescent background with water or stain gel after electrophoresis with EtiBr stain.
Good luck!
Yuri
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