Table 4.

Troubleshooting

Problem	Cause	Solution
Low PCR1 yield	Adapter dimers in PCR1 or low starting quantities	Repeat AMPure XP beads clean-up and/or amplify 5–10 additional cycles. Check the AMPure XP bead calibration.
Low PCR2 yield	Adapter dimers in PCR1 and/or PCR2 or too much salt in the DSN reaction	Repeat AMPure XP beads clean-up on PCR1 and/or amplify 5–10 additional cycles. Check the AMPure XP bead calibration.
Poor rRNA reduction	Adapter dimers in PCR1 or mechanical failure during the DSN reaction	Remove adapter dimers with additional AMPure XP bead clean ups. Mix the DSN hybridization and reaction well, keep the temperature at a constant 68°C during these steps.
No mature miRNA in the smRNA library	Loss during RNeasy isolation of RNA or AMPure XP bead clean-up due to size selection or low ethanol concentration in the bead washes	Use RWT buffer with the proper amount of ethanol for the RNeasy column wash. Check the AMPure XP bead calibration. Make 80% ethanol fresh for RNeasy column and bead washes.
Reduction of rRNA qRT-PCR validation inaccurate	Secondary inflection in the amplification curve resulting in high CT values for the library rRNA amplification	Use the ‘Absolute Quantification Analysis Using Fit Points Method’ to set the cycle count lower for qRT-PCR on the 480 LightCycler.
Poor PCA for biological replicates	Poor size fractionation of the input RNA due to overloading the RNeasy Minelute column	Use RNeasy mini column for the size fractionation or use less RNA. Typically the data will still be robust.