FFPE DNA Extraction
Formalin-fixed, paraffin-embedded (FFPE) tissue samples provide a valuable source of stable nucleic acids for gene expression analysis. The clinical utility of FFPE samples is substantial, where retrospective analysis of archival tissue enables the correlation of molecular findings with the response to treatment and the clinical outcome.
Which DNA extraction kit from FFPE samples is right for you?
|Fast & Easy||Low-throughput isolation of RNA and DNA from the same FFPE section||Scalable isolation of RNA and DNA from the same FFPE section|
|Order now||Order now||Order now|
|Ion AmpliSeq™ Direct FFPE DNA Kit||RecoverAll Total Nucleic Acid Isolation Kit for FFPE||MagMAX FFPE DNA/RNA Ultra Kit|
|Prep time||30 minutes|| 4.5 hours (total time for 16 samples)
2.5 hrs hands-on time
|4 hours (total time for 96 preps)
1 hr hands-on time
|Final product extracted||DNA||total RNA, microRNA, gDNA||total RNA, microRNA, gDNA|
|Isolation method||lysis||column purification||mag bead purification|
|Automation compatible (post deparaffinization)||not applicable||✓
(Scripts for KF Flex and Duo Prime)
|Compatible FFPE sample range||single 5-10 micron section
(4-100 mm2 tissue)
|up to 4 x 20 micron sections
(300 mm2 tissue)
|up to 2 x 20 micron sections
(300 mm2 tissue)
|Sample volume||~20µL lysate containing DNA||~50µL purified DNA
~50µL purified RNA
|~50µL purified DNA
~50µL purified RNA
|Compatible with Ion AmpliSeq DNA panels (e.g. Cancer Hotspot Panel v2)||✓||✓||✓|
|Compatible with Ion AmpliSeq RNA panels (e.g. RNA cancer and RNA fusion)||✓||✓|
Why is nucleic acid isolation from FFPE samples such a challenge?
The fixation process causes cross-linkage between nucleic acids and proteins, and covalently modifies adenine and cytidine by the addition of monomethyl groups to the bases. As a result, the molecules are rigid and susceptible to mechanical shearing. Therefore, in order to utilize FFPE tissues as a source for DNA, a reliable method is required for its extraction from the cross-linked matrix.
How can you increase your chances of success with FFPE samples?
There are a number of factors that can impact the overall quality and yield of DNA isolated from FFPE tissues. Here are recommendations to address several key factors:
- Upstream tissue procurement and tissue specimen preparation—if possible, tissues should be fixed within one hour of surgical resection. The optimal fixation time is 12–24 hours, using neutral-buffered formalin or paraformaldehyde. Fixed tissues should be thoroughly dehydrated prior to the embedding process.
- Block storage—storage of blocks without cut faces, when possible, prevents ongoing damage from exposure to atmospheric oxygen, water, and other environmental factors such as light and infestation (fungus, insects, etc.).
- Tissue type, size, and amount being used for RNA isolation—the recommended tissue thickness is 10–20 µm The number of sections used is determined by the tissue type (which impacts cell density) and surface area (recommended size: 50–300 mm2). Excess starting material can cause filter clogging, resulting in poor yield.
- Excessive amount of paraffin used for embedding tissues—when possible, excess paraffin should be trimmed away prior to starting the purification protocol. For xylene-based purification methods, two xylene treatments at room temperature should be sufficient for complete deparaffinization. If desired, a more rigorous 37–55°C treatment can be performed for up to 30 minutes. After the xylene deparaffinization, it is crucial that the 100% ethanol is completely removed and the pellets are dry after the two 100% ethanol washes. The magnetic bead method employs novel chemistries to deal with the paraffin that limits input to 20 µm sections.
The MagMAX™ FFPE Nucleic Acid (NA) isolation kits are designed for rapid, efficient, and automatable isolation of total RNA and DNA from FFPE samples using MagMAX™ magnetic particles. MagMAX™ FFPE NA kits offer faster workflows and use less toxic reagents, without sacrificing quality in the process. These kits help deliver nucleic acid yield and purity comparable to the best-in-class RecoverAll™ filter-based system. The MagMAX™ FFPE NA kits outperform products with similar protocols, and achieve RNA and DNA yields that are comparable with longer, more hazardous, and lower-throughput protocols.
The RecoverAll™ Total Nucleic Acid Isolation Kit procedure requires about 45 minutes of hands-on time. FFPE samples are deparaffinized using a series of xylene and ethanol washes. Next, they are subjected to a rigorous protease digestion with an incubation time tailored for recovery of either RNA or DNA. The nucleic acids are purified using a rapid glass-filter methodology that includes an on-filter nuclease treatment, and are eluted into either water or the low-salt buffer provided.
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