Final answer:
Poor RNA quality in extractions using the QIAGEN miRNeasy Mini Kit may be due to RNase contamination, omission of the DNase treatment, use of degraded reagents, or improper handling of samples. Ensuring proper technique, using fresh reagents, and including a DNase step might improve RNA quality for downstream applications like RNAseq and qPCR.
Step-by-step explanation:
If you are experiencing poor quality of RNA from your extractions using the QIAGEN miRNeasy Mini Kit - Cat #217004, there could be several factors at play. RNA is highly sensitive to degradation and any contamination with RNases, which are prevalent in the environment, can rapidly degrade RNA samples. Ensuring that all equipment is properly cleaned and that gloves are worn can mitigate this. It is also critical to follow the manufacturer's instructions closely during the RNA purification process.
One step that might be contributing to poor RNA quality is the omission of a DNase treatment. Trace amounts of DNA can interfere with downstream applications like RNAseq and qPCR. The absence of this step could therefore be negatively impacting your results.
Another consideration is the integrity of the reagents used. Make sure that the ethanol and other solutions are fresh and not contaminated. Also, check that your starting material (the biopsies) is processed promptly after collection to prevent RNA degradation. Additionally, after centrifuging, you must properly separate the aqueous phase to avoid DNA or protein carry-over which can also degrade your RNA.
If issues persist after taking these precautions, you may need to perform troubleshooting steps to identify exactly when and where the issue occurs during the purification process. This may involve gel electrophoresis or absorbance measurements to assess RNA degradation and purity at different stages.