Sample prep for cancer research: FFPE tissue
For decades, a standard practice among pathologists has been to obtain tissue biopsies from the tumors of cancer patients, fix them in formaldehyde (also known as formalin), and then dehydrate and embed the fixed tissues in blocks of paraffin wax. This preparation allows for formalin-fixed, paraffin embedded (FFPE) samples to be sliced into thin sections that can be mounted onto microscope slides and subjected to techniques such as immunohistochemistry for further analysis of cell morphology, protein expression, etc. This approach also renders the samples suitable for long-term storage under ambient conditions. As a consequence, clinics and research institutions around the world have accumulated vast repositories of FFPE samples that are paired with relevant patient data. These repositories have proven to be invaluable for researchers seeking to perform large-scale analyses aimed at elucidating the molecular mechanisms of cancer (Fairley et al. 2012).
While biobanking of FFPE tissues has provided cancer researchers with a vital resource, obtaining adequate yields of DNA and RNA of suitable quality for downstream analysis from these samples is not trivial. Fixation results in nucleic acid fragmentation, the formation of crosslinks between nucleic acids and proteins, and the introduction of nucleotide changes, which collectively hinder the performance of downstream assays. A variety of nucleic acid purification methods have been developed specifically for FFPE samples, which yield outputs suitable for applications such as PCR and NGS, to overcome these obstacles.
Please note that Macherey-Nagel nucleic acid purification products are only available from Takara Bio in North America, India, and Japan.
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DNA purification
Takara Bio offers both silica membrane- and magnetic bead-based technologies for the purification of DNA from a wide array of FFPE samples with the NucleoSpin DNA FFPE XS and NucleoMag DNA FFPE kits. Both technologies incorporate the use of an odorless, non-toxic, xylene-free reagent for paraffin removal. Samples are then treated with proteinase to solubilize the fixed tissue and release DNA into solution, followed by heat incubation in a specially formulated buffer to eliminate crosslinks. Lysate DNA is then bound to a silica membrane or paramagnetic beads, subjected to sequential wash steps, and eluted. Whereas the NucleoSpin FFPE DNA kit is provided in both single- and multi-prep formats for varying throughput demands, the NucleoMag FFPE DNA kit has been developed primarily for automated, high-throughput processing.
Figure 1. Outstanding PCR performance due to efficient recovery of de-crosslinked DNA with NucleoSpin DNA FFPE XS. DNA was isolated from FFPE rat liver tissue with NucleoSpin DNA FFPE XS (2x blue graphs) and with an FFPE mini elution kit from Competitor Q (2x orange graphs), and a 100-bp target was amplified by PCR using a Roche LightCycler system. Yields were consistently higher with NucleoSpin DNA FFPE XS, and the DNA provided better PCR performance relative to the output from Competitor Q's kit. For this comparison, the starting sample was one section of FFPE tissue that was subjected to overnight lysis and eluted in a volume of 30 µl.
RNA purification
For purification of total RNA from FFPE samples, Takara Bio offers silica membrane-based technologies in single-prep formats designed for varying input amounts: NucleoSpin totalRNA FFPE and NucleoSpin totalRNA FFPE XS. Following deparaffinization, proteinase treatment, and heating, the lysate is applied to the membrane under conditions that enable binding of diverse RNA species, including small RNAs. Residual DNA is then digested via an on-column DNase treatment and, following successive washing steps, RNA is eluted in a small volume of RNase-free water, yielding highly concentrated RNA.
Figure 2. NucleoSpin totalRNA FFPE provides higher RNA yields relative to competitor kits, as determined by qPCR. Total RNA was isolated from 4 x 10 µm thick FFPE sections of mouse brain tissue with NucleoSpin totalRNA FFPE and three competitor kits (Q, A, and P). mRNA and miRNA targets were amplified by qPCR (the mRNA target consisted of a 230-nt portion of the β2-microglobulin gene, while the miRNA target was analyzed using the miR-16 TaqMan MicroRNA Assay. (Note: miRNA samples were reverse transcribed separately using the TaqMan MicroRNA Reverse Transcription Kit from Thermo Fisher Scientific.) The lower CT values observed for the samples processed using the MN kit suggest that the MN kit provided higher RNA yields relative to competitors' kits.
Reference
Fairley, J. A. et al. Making the most of pathological specimens: molecular diagnosis in formalin-fixed, paraffin embedded tissue. Curr. Drug Targets 13, 1475–1487 (2012).
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