Formalin fixation and paraffin embedding (FFPE) tissues and Fine-needle Aspiration Biopsy (FNA) are popular sources of archival tissue samples that pathologists have used for decades. It enables long-term, room-temperature preservation of tissue samples, making it extremely handy. Because it has been the standard for so long, it is also the primary source of biological research materials. One of the critical steps in molecular oncology diagnostics is obtaining high-quality genomic DNA. Mutation analysis of FFPE- or FNA-derived DNA aids in the diagnosis of most solid tumors. Tests such as Epidermal Growth Factor Receptor (EGFR), Kristen Ras Gene (KRAS), Neuroblastoma Ras Gene (NRAS), and Next Generation Sequencing for significantly mutated genes hot spot detection using FFPE DNA have become essential in determining specific cancer treatment.
However, DNA extraction from these tissues is a challenge mainly owing to formalin fixation, which causes cross-linking between proteins and DNA and distinct DNA strands. As a result, pure DNA is frequently extensively damaged and fragmented. The degree of fragmentation is determined by tissue type, sample age, and specific fixation procedures. These characteristics can cause problems in downstream applications, many of which use PCR. As a result, DNA extraction from FFPE tissues must successfully extract highly fragmented DNA and reverse cross-linking generated by formalin fixation.
To extract enough genomic DNA (gDNA) for downstream analysis, several commercially available nucleic acid extraction kits require a minimum of one 10µm slide of starting material. Because these blocks are acquired from human patients, the extraction kit is critical because it defines the size of the slide needed and the number of analyses performed on each FFPE block. Because some stored tissues are too tiny to meet this condition, this limitation lowers the number of feasible applications. Furthermore, using these kits for DNA extraction might be time-consuming, and the “Bind-Wash-Elute” procedure may cause significant DNA loss.
HygiaReagents One-Step FFPE & FNA DNA Purification Kit is designed to extract total nucleic acids from 2µm – 5µm Formalin-Fixed, Paraffin-Embedded (FFPE) tissue or Fine Aspiration Biopsy samples efficiently and sequentially. The kit employs our unique magnetic beads to efficiently remove paraffin from FFPE tissue samples in an aqueous buffer while simultaneously rehydrating the tissue. The procedure eliminates flammable and odorous xylene or d-limonene and the time-consuming cleanup of organic solvent from frequently hardly visible tissue pellets commonly employed for deparaffinization. Furthermore, the kit is unique because its proprietary magnetic beads remove PCR inhibitors from samples in a single step without needing DNA extraction.
Therefore, it increases nucleic acid yields and avoids DNA loss caused by the time-consuming “bind-wash-elute” procedure used in traditional DNA extraction techniques. Following sample lysis, the straightforward one-step purification technique is ideal for the simultaneous processing of >96 samples and produces pure DNA in less than 30 minutes. Purified genomic DNA has the highest integrity and can be used in various downstream applications such as qPCR, mutation screening, microarray analyses, sequencing, Southern blotting, and SNP analysis.
Workflow Of One-Step FFPE And FNA DNA Isolation And Results
1. To lyse the sample, add functional magnetic beads and proteinase K to the sample and incubate at 65°C.
2. Vortex/pipette the beads with the sample to capture the PCR inhibitors.
3. Separate the beads from the sample using a magnet.
4. Aspirate the supernatant containing the pure, ready-to-use DNA/RNA.
Features and Advantages
● Rapid and efficient isolation protocol: without prior DNA isolation for subsequent use in direct workflows, No liquid transfer, and One-tube.
● Ultrafast: Process 96 samples in less than 30 minutes.
● Highest nucleic acids recovery rates: Minimal loss of DNA during extraction.
● Effectively cell lysate cleanup and removes inhibitors: polyphenolic compounds, humic/fulvic acids, acidic polysaccharides, tannins, melanin, heparin, detergents, denim dyes, divalent cations such as Ca2+, Mg2+, etc.
● Highly improved active paraffin removal: No need for toxic organic solvents, such as xylene.
● Cost-effective: Eliminates columns, filters, laborious repeat pipetting, and organic reagents.
● High-throughput: Compatible with many different automated liquid handling systems. |
