Dewaxing Solution

Although the RIN number can provide general information about the extent of sample fragmentation, it is not sensitive or predictable enough to be a useful indicator of downstream performance, especially for RNA-Seq. Very often, RIN numbers for FFPE-derived RNA will be between 2 and 3. Some of these samples will be useful for RNA-Seq, and others won’t – the RIN will not tell you, however.

A slightly better predictor of performance in RNA-Seq using Illumina sequencing is the DV200, which represents the percentage of RNA fragments longer than 200 nucleotides. The DV200 is also calculated based on Bioanalyzer data, but suffers from the same drawbacks as all Bioanalyzer-based methods, specifically high variability.

• Avoid methods based on organic solvents (Trizol)
• Avoid harsh chaotropic salts (i.e. guanidinium)
• Avoid detergents that impact downstream quantitation by UV and/or Qubit (e.g. Triton X-100)
• Do not rely on RIN to quantitate the integrity of an FFPE-derived sample, use DV200 instead.
• Use a kit or method that removes chemical modifications from formalin. Do not raise the temperature to 80˚C or above. Even short times at this temperature will significantly lower integrity.
• Be wary of Qubit and Nanodrop concentrations because of the possibility of contamination by organic molecules or DNA.
• Use qPCR to quantitate your RNA, and always look carefully at melt curves to determine whether nonspecific amplification may have occurred.

Due to the wide size distribution of DNA isolated from FFPE tissue samples, we recommend using pulsed-field gel electrophoresis (PFGE). Methods based on capillary electrophoresis such as the Agilent Bioanalyzer can also be used but may not properly resolve high molecular weight fragments (greater than 10kb) in better-quality samples.