Chablina Hazarika Despite good precautions and A260/280 ratios, smearing on gel with no distinct 28S/18S rRNA bands suggests possible RNA degradation during or after extraction, likely due to residual RNase activity in plant tissues, delayed TRIzol inactivation, or issues during phase separation or isopropanol precipitation. Ensure TRIzol is added immediately after grinding (not delayed), use fresh TRIzol, avoid over-drying the RNA pellet, and consider adding β-mercaptoethanol during lysis to inactivate stubborn RNases. Additionally, check that gel loading buffer and gel itself are RNase-free, and try using a bioanalyzer or TapeStation for more precise integrity assessment (RIN).

Dear Chablina,

As Rajarethinam said, test your RNA on Bioanalyzer. Gel by itself can make results looking worse than they are in reality. If Bioanalyzer will support gel, then you can think about improvements of the collection/extraction process.

In the final step, after elution, incubate the RNA at 50 °C for 10 minutes. Give it a brief vortex and spin down. Before that, during the drying step, make sure the ethanol is fully evaporated. In my experience, residual ethanol can cause issues during gel visualization. In this case, you can also check the A260/230 ratio. If it’s around 1.4, it likely indicates the presence of residual ethanol. Hope this helps!