RNA Preparation Troubleshooting

It is good practice to check RNA quality, purity, and yield before using prepared RNA in a downstream application. Troubleshooting downstream applications may require that you go back to the prepared RNA sample. First, check RNA quality and purity using RIN, the 28s:18s ratio, and the A₂₆₀/A₂₈₀ ratio, as discussed above. If the RNA quality is poor, this is probably the cause of poor results in downstream applications. In this case, either RNA was degraded before the sample was processed or RNA was degraded during preparation. The RNA preparation should be repeated with fresh samples and with more care to prevent RNase contamination during the procedure. If the quality of RNA is good, check the yield of RNA. If the RNA quality is good but the yield is lower than expected, this might be due to incomplete cell lysis, poor binding, or poor elution if using silica purification. The RNA preparation should be repeated with more care to make sure the sample is completely disrupted and the cells completely lysed. If the RNA yield is lower than expected, RNA may need to be concentrated, or more RNA may need to be prepared (e.g., increase sample input). If quality, purity, and yield of RNA are good, the problem may be with the downstream application (Table 1).