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Table 5 Troubleshooting advice

From: Detection and quantification of extracellular microRNAs in murine biofluids

Step Problem Solution
1-5 Insufficient volume collected Biofluid must be collected instantly post-mortem to avoid clotting related problems. The angle of the collection tube is critical. Avoid getting blood on the outside of the collection tube as the surface tension will allow the sample to flow back out of the tube.
  Hemolysed sample Avoid vigorously mixing or shaking blood collection tubes. Minimize contact of blood with water or exposure to heat and ensure that samples are kept upright. Do not freeze blood samples before separation.
6-29 Low RNA yield RNA yield is expected to be very low (~0.1 ng/lμl). Improved yield may be obtained by increasing biofluid volume (scale reagents accordingly). Increasing precipitation time and including the optional re-hydration of phenol phase and MS2 RNA carrier steps can also improve RNA recovery. Re-suspending the RNA pellet in a smaller volume increases concentration but may decrease yield.
43-50 No amplification May be a real result. Include a positive control to verify.
  Atypical amplification curve Adjust fluorescent background. If the shape of the curve does not improve exclude effected wells.
  Atypical melt profile (if applicable) Exclude wells. Melting artefacts may occurs at low copy numbers or be caused by sub-optimal assay design. Switch to a probe-based detection technology or re-design assay.
  Template inhibition Exclude samples over the range where inhibition is observed. Dilute samples (and therefore also PCR inhibitors) and re-run qPCR.
  Amplification in NTC or RT- reactions Indicates reagent contamination. Switch to new reagents and clean work surfaces.
  Low PCR efficiency Some assays may exhibit sub-optimal PCR efficiencies due to restricted target space. Improvements can be obtained by optimizing annealing temperature in the PCR protocol. Sub-optimal PCR efficiencies can be used with caution. Consider re-designing assay or switching to an alternative detection technology.