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.