About ZR FFPE DNA MiniPrep™
The ZR FFPE DNA MiniPrep™ provides a simple and reliable method for high yield/quality DNA isolation from formalin-fixed, paraffin embedded (FFPE) tissue samples and sections. The unique chemistries of the product have been optimized for maximum recovery of non-crosslinked, ultra-pure DNA without RNA contamination. Simply digest deparaffinized tissues using the provided Proteinase K, heat, and then purify the DNA with the Fast-Spin columns in the kit. DNA <50 bp or <500 bp can be selectively isolated by altering the lysis buffer conditions as given in the protocol. PCR inhibitors are effectively removed during the isolation procedure, and eluted DNA is ideal for PCR, Next-Gen library prep, enzymatic manipulation, etc. Shown below is a schematic and performance overview of the procedure.
|DNA Recovery||High quality total DNA (A260/A280 >1.8) can be eluted into small volumes (i.e., ≥25 µl) allowing for highly concentrated samples. The maximum DNA binding capacity of the provided spin column is ~25 µg.|
|Processing Time||As little as 4 hours when processing large amounts of tissue. For maximum yields of the highest quality DNA, it is recommended to process samples overnight.|
|Sample Size||Up to 25 mg tissue from paraffin block or up to four (4) tissue sections (≤20 µm thick) with a total surface area ~20 cm2. It is recommended to use 1-2 sections if performing the protocol for the first time. Compatible with fresh/frozen tissue specimens.|
|Equipment/Reagents||Microcentrifuge, thermomixer or heat block/bath capable of 55°C and 90°C, xylene, ethanol, isopropanol, beta-mercaptoethanol (optional).|
To a deparaffinized tissue sample (≤25 mg) in a microcentrifuge tube, add the following:
Note: If your tissue sample is too large for the digestion volume, scale up the digestion to 200 µl while keeping the amount of Proteinase K the same. Double the reagent volumes indicated in Step 3 & 4.
Note: The Rapid Digestion is recommended for processing slide tissue sections but should also besufficient for most tissue samples. However, the Standard Digestion ensures maximum yields of DNA from even tough-to-lyse (collagen-rich, fibrous, etc.) tissue samples.
Transfer the digestion to 94°C and incubate for 20 minutes. When done add 5 µl of RNase A, mix, and then incubate an additional 5 minutes at room temperature.
Note: It is recommended to skip the heat treatment in Step 3 and the addition of isopropanol in Step 4 if only double stranded DNA is required (e.g. OneStep qMethyl™ Array, Cat. No. D5312).
Add 350 µl of Genomic Lysis Bufferto the tube and mix thoroughly by vortexing.
Note: When working with a new sample, it is recommended to isolate total DNA as a precaution. FFPE DNA may be highly degraded and DNA >500 bp may not be present in sample.
Centrifuge at 10,000 x g for 1 minute to remove insoluble debris and then transfer the supernatant to a Zymo-Spin™ IIC Column in a Collection Tube. Centrifuge at 10,000 x g for 1 minute.
Add 200 µl of DNA Pre-Wash Buffer to the spin column in a new Collection Tube. Centrifuge at 10,000 x g for 1 minute.
Add 400 µl of g-DNA Wash Buffer to the spin column. Centrifuge at 10,000 x g for one 1 minute.
Transfer the Zymo-Spin™ IIC Column to a clean microcentrifuge tube. Add ≥50 µl DNA Elution Buffer or water (e.g., add ≥100 µl if sampling 25 mg tissue) to the spin column. Incubate 2-5 minutes at room temperature, then centrifuge at top speed for 30 seconds to elute the DNA. The eluted DNA can be used immediately for molecular based applications or stored ≤-20ºC for future use.
Yun et al. used the ZR FFPE DNA MiniPrep™ kit to purify high quality DNA from formaldehyde-fixed paraffin-embedded (FFPE) tissues for subsequent analysis by mass spectrometry, and they found that this kit was superior to other DNA purification methods. The authors then detected DNA that was covalently modified by toxic chemicals, allowing them to monitor exposure to potentially carcinogenic substances. Use of the ZR FFPE DNA MiniPrep™ kit allowed this novel detection method, which was previously limited to fresh tissues, to be used to investigate collections of FFPE tissues for carcinogen exposure, thus permitting discovery new cancer biomarkers and epidemiological studies of cancers with environmental etiologies.