mirVana™ miRNA Detection Kit - FAQs

With the mirVana miRNA Detection Kit, I am getting aberrant migration of my miRNA with pointed or smeared bands. Why is this?

Here are possible causes and solutions:

1. Residual salt in the RNA pellet: if the supernatant from the final precipitation step is not completely removed, salts from the precipitation mixture will be concentrated and may lead to aberrant migration (“tunneling”) of the protected fragment. We recommend briefly respinning the final pellet after the supernatant is removed, and then thoroughly aspirating any residual supernatant using a very fine-tipped pipet. Alternatively pellets can be washed with 75% ethanol.
2. Gel quality and sample volume: for best resolution, we recommend letting the gel polymerize for at least 1 hr and pre-running the gel for at least 1 hr at constant current prior to loading. It is also critical to rinse the urea out of the wells immediately before loading. Finally, overloading gel wells reduces product resolution and may affect data quality. Wells that are 0.4-0.6 cm wide work well for loading the 5-10 µL samples from this procedure. The best resolution is obtained when the gel loading buffer forms a 2-3 mm layer in the well.
3. Radioactive material that fails to migrate into the gel: it is unclear what causes this problem; it may be the result of several different factors. Probes that have not been gel purified exhibit this “hang up in the well” phenomenon more frequently than gel-purified probes. Phenol/chloroform extraction of probes that were not gel purified tends to diminish the amount of material left in the wells. Another contributing factor seems to be residues left in microcentrifuge tubes from the manufacturing process. Autoclaving of siliconized tubes appears to exacerbate this problem.