Dewaxing Solution

The RNAstorm™ FFPE and DNAstorm™ FFPE extraction kits include a recommended Deparaffinization Reagent. Unlike other common methods (e.g., xylenes), the Deparaffinization Reagent is efficient, non-toxic, and does not require the use of a fume hood. In our testing, the included reagent is at least as effective as xylenes at removing paraffin and allowing purification of high-quality nucleic acids.

FFPE-derived RNA is much more challenging to quantitate accurately than RNA obtained from fresh samples. It is not enough to know the absolute amount of RNA that is present, but also whether the RNA will work in downstream applications, which depends on the following factors:

• Fragment size distribution: a 5 µg sample (as measured by Qubit) can be useless for RNA-Seq if it consists of fragments < 200 nt.
• Chemical modification: for RNA obtained from formalin-fixed samples, various chemical adducts and crosslinks, including base modifications, base-base crosslinks, and base-protein crosslinks can make nucleic acid molecules inaccessible to enzymes and therefore inactive in downstream applications.
• Contamination: cellular debris, proteins, salts, and detergents used during purification can bias downstream assays. For example, UV/Vis-based methods such as Nanodrop are particularly susceptible to contaminants that absorb in the 200-280 nm range.
• Fluorescence-based methods such as Qubit are liable to significant error. When working with low concentrations of DNA or RNA, dye-based detection may not be linear. One must also be mindful of contamination by genomic DNA in an RNA sample because the dyes used for fluorescence quantitation are not entirely specific for FFPE-derived DNA or RNA.
• Quantitative PCR is the preferred method for quantitation of heavily damaged and modified nucleic acids.

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.