Archived Formalin-fixed, Paraffin-embedded (FFPE) tissue samples are invaluable resources for profiling gene expression and studying a variety of diseases. Since the archived DNA is usually available in limited quantities, amplification of the samples is essential. Amplifying the FFPE tissue can be a difficult task due to the damaged template that results from the archiving process. This protocol provides a convenient method to purify and amplify genomic DNA from FFPE tissue. GenomePlex® Whole Genome Amplification has been used to amplify genomic DNA from rat and human FFPE tissue samples.
Note: The protocol below describes the process of DNA purification and subsequent whole genome amplification. We have dramatically streamlined this process with our GenomePlex® Tissue Whole Genome Amplification Kit (Product No. WGA2+C4482). This kit includes optimized reagents for tissue disruption and cell lysis eliminating the need for tedious organic extractions to remove excess paraffin or DNA purification prior to amplification.
Extraction of DNA from Formalin-Fixed, Paraffin-Embedded (FFPE) Tissue
This protocol was performed on rat liver tissue. The GenElute™ Mammalian Genomic DNA Miniprep Kit (G1N10) is recommended for this procedure.
GenomePlex® Whole Genome Amplification FFPE Tissue
Legend: Products were amplified using the GenomePlex® Whole Genome Amplification Kit (WGA1) from us, Supplier A, and Supplier Q. Products were resolved on a 1.5% agarose gel. 5 µL of amplified product was added to each well. The products amplified using GenomePlex technology were of a smaller molecular weight as shown on the gel when compared to Supplier A and Q. This is due to the random fragmentation of genomic DNA prior to amplification. Our amplified products are specific and there is no amplicon visible in the negative control (lane 2) indicating that only the desired genomic DNA is amplified. Both Suppliers A and Q yield a nonspecific signal in the negative control which is equal in size and intensity to the signal for the suppliers’ positive control.
|Lane 1—1 kb Marker||Lane 6—Supplier A 10 ng input DNA|
|Lane 2—Our Negative Control||Lane 7—Supplier A 100 ng input DNA|
|Lane 3—Our 10 ng input DNA||Lane 8—Supplier Q Negative Control|
|Lane 4—Our 100 ng input DNA||Lane 9—Supplier Q 10 ng input DNA|
|Lane 5—Supplier A Negative Control||Lane 10—Supplier Q 100 ng input DNA|
Legend: DNA was extracted from a sample of formalin-fixed, paraffin-embedded rat liver. 10 ng and 100 ng of genomic DNA was amplified using GenomePlex® Complete WGA Kit (Product No. WGA2) followed by purification using the GenElute™ PCR Clean-up Kit (NA1020). Quantitative PCR (45 cycles) was performed on the WGA reaction using 12 different primer sets. GenomePlex demonstrates ~1000 fold (10 Ct) better representation compared to suppliers.
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