Hi Serena,

This is an interesting question. Since you stated that you are following the protocol for the system, I suggest the following.

Focus on thorough sample disruption and homogenization, ensuring proper lysis conditions, using the recommended sample input amount, avoiding excessive mechanical disruption, and carefully following the elution protocol using a larger elution volume if necessary. Make sure you always maintain RNase-free conditions throughout the process. Heating the elution buffer to the recommended temperature for better RNA release would be best. I always evaluate the quality of my RNA using a bioanalyzer or agarose gel electrophoresis to ensure RNA integrity before proceeding with downstream applications. I hope this helps.

Have a look at the following document: Using nanodrop you might be able to identify what contaminants you have in your sample;

https://assets.thermofisher.com/TFS-Assets/CAD/Product-Bulletins/T123-NanoDrop-Lite-Interpretation-of-Nucleic-Acid-260-280-Ratios.pdf

It might be difficult to troubleshoot the exact problem without knowing the contaminant, but the kit likely has washing step(s) in the protocol. My initial thought is to repeat the washing step 2-3 times (sometimes indicated as optional), which can make quite a difference.

If the buffers from the kit are shared with other lab members, it is also possible the kit is contaminated, sometimes stored incorrectly, or too old. You can repeat the experiment with entirely new buffers and reagents as a last resort.

Poor homogenization leads to poor yield. A magnetic bullet blender efficiently homogenizes samples. Alternatively, liquid nitrogen can also be used to pulverize samples with a mortar and pestle. Although an older method, it remains effective in certain cases.