As my experiment involves RNA isolation, I just want to quickly check the total RNA integrity on a gel and confirm it on a UV-spectrophotometer. For that purpose, is a non denaturing agarose gel of 1-1.2% in TBE buffer a good option? Also a final volume of 2ul ethidium bromide is sufficient or should I add more for viewing RNA? I have seen from other sources that ethidium bromide amount is added more than usual unlike what is used for DNA. And is EtBr a good chemical to visualize RNA under UV? Kindly input your valuable suggestions.
PS: I am planning to incubate the RNA samples at 70C waterbath for 3 mins and place them on ice before loading on to the gel. Also my lab does not have an Agilent Bioanalyzer which I am sure is the best solution for RNA quality analysis but please suggest some possible solutions for agarose based method as well.
Thanks
we use non denaturing gels but the sample should be denatured into loading buffer 80% desionized formamide final concentration, EDTA, bromophenol blue) for 10 minutes at 70°C
Reagent Quantity (for 10 mL of 1.5×) Final concentration
Formamide, ultrapure 9.5 mL 95%
Bromophenol blue (2.5%, w/v) 100 µL 0.025%
EDTA (0.25 m, pH 8.0) 200 µL 5 mm
Use 5 µL for a 2.5-μL sample. Purchase a distilled, deionized preparation of formamide and the above loading dyes. Store in small (1-mL) aliquots for up to 1 yr at −20°C. This solution is available commercially (Ambion) and is recommended over homemade.
Hello Achintyan,
Yes, non-denaturing agarose gels can be used. However, the interpretation of results can prove difficult. In non- denaturing gels, the secondary structure of RNA alters its migration pattern and does not migrate according to its actual size. The RNA bands are not sharp and sometimes multiple bands representing different structures of a single RNA species show up. Due to above mentioned shortcomings, it is advisable to electrophorese RNA on a denaturing agarose gel.
However, if you are only looking to see if your RNA is intact or not, then a non-denaturing gel is fine. I often use one to run a couple of microliters of RNA out just to see if I've isolated RNA of good quality or if there is any smearing which would indicate that the RNA is degraded and to what degree. Also, I add the EtBr to my gel, so for a 100ml agarose solution I would add 10ul of EtBr after microwaving, I've never need to add ore EtBr than what I use for DNA. The only thing I do to try and keep everything as clean as possible is to wash the gel box and the comb out with RNAse away and rinse with millipore water. This is the quick and dirty way to quickly access the quality of your RNA. If you want to see if you have the right size band though, this won't work, and you will need a denaturing gel for that.
RNA is very unstable and it won't be good idea to heat it at 70C in water bath. For checking RNA integrity, 0.8% agarose gel is fine with EtBr. But the main thing is that you need to have RNAse free electrophoresis, TAE/TBE buffer and other apparatus. Adding a few microliters of RNAse inhibitor in running buffer gives excellent visualization of RNA.
we use non denaturing gels but the sample should be denatured into loading buffer 80% desionized formamide final concentration, EDTA, bromophenol blue) for 10 minutes at 70°C
Reagent Quantity (for 10 mL of 1.5×) Final concentration
Formamide, ultrapure 9.5 mL 95%
Bromophenol blue (2.5%, w/v) 100 µL 0.025%
EDTA (0.25 m, pH 8.0) 200 µL 5 mm
Use 5 µL for a 2.5-μL sample. Purchase a distilled, deionized preparation of formamide and the above loading dyes. Store in small (1-mL) aliquots for up to 1 yr at −20°C. This solution is available commercially (Ambion) and is recommended over homemade.
Agree with the above, and would only add that we have had good success with adding bleach as per the protocol described by Jorcyk et al in the link, below.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3699176/
I would recommend using a different stain than EtBr. Its high background because of only a moderate (~25x) increase in intensity when bound to nucleic acid, makes it a poor stain for ssNAs, even under non denaturing conditions. Sybr gold or something similar would be far better.
Why not just run a denaturing ( formaldehyde-MOPS) agarose gel or TBE urea PAGE? you will get much nicer/more Interpretable results with only a small amount of extra effort.
u can use non-denaturing agarose gels, i would like to suggest, make your buffer in DEPC treated water, and run the gel at 150V for 10 min. As RNA is very unstable, visualize the gel quickly after run
Thank you all for your valuable suggestions. I used non-denaturing agarose in TAE buffer and incorporated household bleach in it as used in the paper suggested by Dr Matthew Hayden. I was able to get good results with the method.
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