MICROBEnrich™ 试剂盒

Here are possible causes and solutions:

1. Input total RNA is degraded: to see whether the RNA used in the MICROBEnrich procedure was degraded from the start, evaluate a 0.5-5 µg sample of the input RNA mixture on a denaturing agarose gel (see section III.C starting on page 15 of the manual [https://tools.thermofisher.com/content/sfs/manuals/cms_057050.pdf]). Total RNA should produce rRNA bands that appear sharp and well-defined (Figure 3 on page 16). In high-quality RNA samples, the 28S rRNA band will be 1.5-2 fold brighter than the 18S rRNA band.
2. Practice RNase-free technique: all of the typical precautions against RNase contamination should be observed. Gloves should be worn at all times and changed frequently to avoid the introduction of RNases. Bags containing the centrifuge tubes and the solution tubes and bottles should be kept closed when they are not in use to avoid contamination with dust. Any tubes or solutions that will contact the RNA (that are not supplied with the kit) should be free of RNases.
3. Degradation caused by the heat denaturation step: RNA degradation can occur at elevated temperatures when there are divalent cations in the solution. If your RNA solution does not contain at least 1 mM EDTA, contaminating divalent cations could cause RNA hydrolysis during the heat denaturation step (step II.B.3 on page 8).