Final answer:
Denaturing PAGE is a technique used to separate RNA molecules by molecular weight. Urea is used to denature RNA, ensuring that it unfolds and that separation is based solely on molecular weight. Denaturing PAGE provides cleaner RNA isolation for advanced scientific applications.
Step-by-step explanation:
Denaturing PAGE
Denaturing polyacrylamide gel electrophoresis (denaturing PAGE) is a methodology primarily used for separating RNA molecules by molecular weight. RNA is visualized using ethidium bromide (EtBr) staining after electrophoresis. Denaturing PAGE works similarly to agarose gel electrophoresis used for DNA, where negatively charged RNA migrates from the negative to the positive electrode. However, two key differences are:
- The separation medium uses polyacrylamide instead of agarose, which allows for finer separation of smaller nucleic acids due to its smaller pore size.
- RNA samples are denatured using chemicals like urea, which is a chaotropic agent that competes with nucleobases for hydrogen bonding, leading to the unfolding of the RNA molecule. This denaturation ensures separation based solely on molecular weight, as it prevents RNA secondary structures from influencing migration rates.
Denaturing PAGE provides the cleanest RNA isolation, critical when high-purity RNA is required for applications such as structure determination or time-resolved fluorescent spectroscopy. Making a denaturing PAGE gel involves pipetting a urea gel mixture into the assembly for electrophoresis. This method removes impurities like the RNA polymerase, DNA template, unused nucleotides, and degraded RNA fragments.