Table 1.

Troubleshooting commonly encountered problems.

Problem	Possible Cause	Solution
Starting mRNA integrity poor	Obtained material has been degraded, or purification protocol suboptimal, RNase contamination of material	Verify starting material again by Bioanalzyer or formaldehyde gel. If necessary, purchase new material or freshly purify from cellular origin
mRNA size distribution after fragmentation smaller than desired (< 200 nt)	Excessive hydrolysis caused by extended incubation at 94 °C with Mg2+ fragmentation kit	Reduce incubation time at 94 °C. Compare several different incubation times followed by size distribution analysis.
mRNA pellet not visible after precipitation	Omission of glycogen	Ensure addition of glycogen co-precipitant when performing EtOH precipitation of RNA, particularly when trying to precipitate small RNAs at low concentration.
mRNA pellet will not dissolve after glyoxalation	Excessive glyoxalation or overdrying of pellet after precipitation	Reduce glyoxal treatment time or limit drying step after EtOH precipitation. Hydrate pellet and leave undisturbed for 30 minutes or longer. Brief heating (30–60 second intervals) at 95 °C can also be used to aid pellet dissolving.
mRNA size distribution smaller after EndoVIPER	RNase degradation or chemical hydrolysis of material	Check integrity of all components used in assay and remake or order new items if needed. Additional RNase inhibitor may also help.
No detectable mRNA after EndoVIPER	Degradation or hydrolysis of material, loss of material during precipitation or purification steps	See above notes. Check integrity of all components. Ensure use of glycogen and practice care during precipitation and purification steps.