I need to size dsRNA on agarose gel. I am wondering if I can use a DNA ladder. I was looking at the dsRNA ladder from NEB but the largest band is only 500 bp. However, my gene is supposed to be greater than 750 bp.
Hello Alice, Thanks! I actually used the recipe from NEB. Here is the link: https://www.neb.com/products/b0363-rna-loading-dye-2x. My adapted recipe is as follows: 485 uL of RNase free H20, 480 uL of >99.5% formamide, 25 uL of 0.5 mM EDTA (I made a 2mM EDTA in 1mL RNase free H2O first and added 10 uL of B-ME), 10 uL of 0.01% SDS and Bromophenol Blue (I made 1% SDS and Bromophenol Blue in 1 mL of RNase free H2O first). It says you can use it for both denaturing and non-denaturing gels. For non-denaturing gels, you just don't heat the samples. To be safe I kept my samples on ice after adding the loading dye. At first I was a bit suspicious as it contained formamide. However, I ran my first gel yesterday using the loading dye and it didn't denature my dsRNA.
I was doing RNase 1f digestion so basically the RNA that I was analyzing was double stranded. I did run control lanes but I didn't heat the samples. But I can say that the dsRNA lanes were sharp. Also I used the same loading buffer for the dsRNA ladder and I was still able to size it.
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