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).

Table 1. Possible causes and solutions for problems that may occur during RNA preparation.

Problem	Possible cause	Solution
RNA is degraded/is of low quality/purity (e.g., low RIN value, low 28s:18s ratio, or smearing on gel, low A260/A280 ratio [< 1.9])	Starting sample was not stored/handled properly.	Endogenous RNases may have degraded the RNA. Follow recommendations for storage and handling of the sample. Quickly add sufficient chaotrope or denaturant to inactivate RNases in sample. Do not let sample thaw if it is already frozen.
	RNases were introduced during/after sample processing.	Make sure to wear gloves at all times and to change them frequently. Follow other laboratory procedures for treating glassware, plasticware, and solutions that will come into contact with RNA.
	Purified total RNA was not stored properly.	Follow recommended storage conditions to minimize degradation. Store RNA at -80 °C, especially for extended periods.
Total RNA contains genomic DNA	DNase treatment was not used during preparation or was insufficient.	Treat with DNase. Be sure to completely remove DNase after treatment. For silica purification, if an on-column DNase digestion will be done, washing steps will remove the DNase. Alternatively, perform a phenol/chloroform extraction, precipitate the RNA, and resuspend in RNase-free water.
	Purification system was overloaded.	Follow sample size recommendations for your purification system.
Yield of total RNA is low	If the yield is low but quality is good:
	The starting material has low RNA content or the wrong starting material was used (i.e., used some connective tissue instead of the target organ). For silica methods: Elution was not effective.	Use the correct starting material or more starting material. Do not overload the purification system. Apply at least 50 μL of water to the center of the column.
	If both yield and quality are low: Starting sample was not stored/handled properly.	Endogenous RNases may have degraded the RNA. Follow recommendations for storage and handling of the sample. Quickly add sufficient chaotrope or denaturant to inactivate RNases in sample.
	RNases were introduced during/after sample processing.	Make sure to wear gloves at all times and to change them frequently. Follow stringent laboratory procedures for treating glassware, plasticware, and solutions that will come into contact with RNA.
	Sample was not disrupted/ homogenized thoroughly.	Follow disruption/homogenization recommendations for your sample type.
Total RNA performs suboptimally in downstream application	If RNA quality is poor:	See troubleshooting tips for poor-quality RNA.
	If RNA quality is good: Contaminants may still be present in the sample.	Do not overload the purification system. This may result in poor yield and/or decreased purity of total RNA. If organic solvents were used, residual solvent may be present in the sample. Precipitate the RNA with salt and alcohol. For low concentrations of RNA, add an inert coprecipitant (e.g., glycogen).
	For silica purification using ethanolic wash buffer: residual ethanol remains in the sample.	Be sure to thoroughly air dry (e.g., extra spin) the sample prior to elution.
	Genomic DNA is present.	See troubleshooting tips above.
For mRNA: Sample contains a significant amount of rRNA	Sample was not disrupted/lysed thoroughly.	Follow disruption/lysis recommendations for your sample type.
	oligo(dT) purification system was overloaded.	Follow sample size recommendations for your purification system. Sample may require a second round of oligo(dT) chromatography.