RNeasy FFPE Kit From Qiagen

Tissue samples in basic research studies of both plants and animals are often modified chemically to preserve biological integrity and allow for long-term storage. However, nucleic acids present in samples that are formalin fixed and paraffin embedded (FFPE) are extensively cross-linked with other cellular components, complicating the extraction process. Qiagen is one of several companies that offer an RNA extraction product (RNeasy FFPE Kit) specifically designed for isolating total RNA from FFPE samples. Proprietary reagents in the kit reverse formaldehyde modifications and allow extraction of fragmented mRNA molecules that can then be assayed using qPCR and modified microarray based methods.

The RNeasy FFPE system is designed similarly to Qiagen’s other RNA extraction kits, making the transition to extraction from FFPE easy for those with previous experience. For the first-time user, the protocols and the principles underlying each step of the extraction process are well described within the user manual. The RNeasy FFPE kit utilizes a brief thermally conditioned enzymatic digestion to optimize lysis conditions and includes QIAGEN’s patented system for removing genomic DNA. Thus, there is no requirement for DNase digestion or overnight incubations, which tend to reduce RNA yields.

To extract total RNA from cells in FFPE samples, 5-10 um tissue sections or cells isolated by laser microdissection from tissue sections, are first subjected to a short incubation at high temperature in a lysis buffer containing proteinase K to reverse the formalin crosslinking of nucleic acids. The cell extract is then passed over a gDNA spin column to remove genomic DNA. The flow through is retained for the next step where it is mixed with ethanol to allow the RNA to bind to Qiagen’s standard RNeasy MinElute spin column. After binding, the RNA is washed and then eluted with deionized RNase free water (supplied). The mRNA species in then eluted; total RNA can then be amplified linearly and assayed to determine mRNA levels using qPCR or specialized microarray platforms (e.g. Affymetrix X3P GeneChip™ arrays).

Our lab has utilized the RNeasy FFPE Kit to extract total RNA from laser captured epithelial and lymphoid cells isolated from human and non-human primate tissue samples fixed in 10% formalin over a range of incubation periods. We have found that RNA can be extracted efficiently, even in samples subjected to longer fixation periods (>24 hr). Although the difference in RNA yields between samples fixed for less than 24 hr versus those fixed for longer periods was not as great as expected, we have been impressed with the ability of this extraction system to yield amplifiable mRNA from our most “highly fixed” sources. The total RNA extracted in these studies has been amplified and labeled successfully for subsequent hybridization to Affymetrix GeneChips™. Moreover, the isolated RNA was also amendable to assay by real-time quantitative PCR and produced data supporting the array analysis.