Hi everyone,

I've been trying to replicate the protocol described in this paper: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22736012/ for separating aminoacyl-tRNAs from deacylated tRNAs using denaturing acid PAGE. Briefly, the protocol involves RNA extraction using TRIzol, followed by RNA suspension in an acid buffer (sodium acetate 10mM pH 5.0, EDTA 1mM pH 8, Urea 8M), and separation of RNA species using Acid-urea polyacrylamide gel electrophoresis (6% or 12% acrylamide solution, sodium acetate 100mM pH 5.0, EDTA 1mM pH 8, Urea 8M).

Despite following meticulously the protocol (including using the same apparatus as the authors), I'm encountering some issues. Specifically, both the RNA and the two tracking dyes I'm using are migrating much slower than indicated, even after running electrophoresis for up to 24 hours and experimenting with increased voltage (ranging from 100V to 200V). Instead of distinct separation, I'm observing minimal to no separation of the dyes and very small RNA migration.

I've also attempted substituting sodium acetate and EDTA with TBE 1x while maintaining the same urea concentration, as described in this other protocol I found: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8848930/. However, the same issues persist.

Lastly, I've noticed fluctuations in the current output of the generator during the run, unlike my experiences with SDS-PAGE for proteins or TAE-agarose gels for DNA/RNA separation. I'm unsure if this is a cause or a consequence of my problems, or if it's normal for this type of acid polyacrylamide gel electrophoresis.

Could someone kindly advise on potential solutions? Should I consider further increasing the voltage and extending the duration of the run?

Thank you in advance,

Giorgio