For decades, FFPE tissue has been the staple of histologists and pathologists for creating tissue slides and making patient diagnoses.
More than 400 million FFPE tissue samples exist for cancer studies alone, and many of these samples can be tied to clinical data. Using one simple kit, the RecoverAll™ Total Nucleic Acid Isolation Kit (patent pending), researchers can now isolate total nucleic acids (DNA and RNA, including miRNA) from the same FFPE sample. Efficient protease digestion conditions allow RNA isolation in less than one day. The nucleic acids recovered are ideal for various downstream applications such as microarray analysis, qRT-PCR, and mutation screening.
Nucleic Acid Isolation from FFPE Tissue
Total RNA Isolation from FFPE Samples
Gene Expression from FFPE Samples
Figure 1. Ct Values from Real-Time RT-PCR of Frozen and FFPE Mouse Brain. Half of each of four mouse brain samples were flash frozen in liquid nitrogen then stored at –80ºC; the other half was fixed and embedded using a standard hospital protocol. RNA was isolated from one 20 µm slice from each FFPE mouse brain using the RecoverAll™ Total Nucleic Acid Isolation Kit. RNA from the frozen controls was isolated using the mirVana™ miRNA Isolation Kit. RNA (400 ng) from each sample was used in two-step real-time RT-PCR. cDNA was synthesized using the RETROscript® Kit. Random decamer primers, one tenth of the RT reaction, and SuperTaq™ Polymerase were used for PCR. Each bar represents the mean ± standard deviation for 8 replicates. TBP=TATA Binding Protein; GAPDH=Glyceraldehyde-3-phosphate Dehydrogenase; UBC=Ubiquitin C; PKC=Protein Kinase C; Recc1=Replication Factor C; RNAPol II=RNA Polymerase II; FAS=Fatty Acid Synthase; DDPK=DNA Activated Protein Kinase.
DNA Isolation from FFPE Tissues
Figure 2. Agilent® 2100 bioanalyzer Scan Demonstrating Presence of RNA and DNA from FFPE Mouse Liver. Nucleic acids were isolated from FFPE mouse liver (20 µm section, fixed and embedded using standard hospital protocol) using the RecoverAll™ Total Nucleic Acid Isolation Kit. Three equal amounts of sample (based on A260) were treated as follows: untreated control received no DNase or RNase treatment; RNA was isolated by DNase treatment of the nucleic acid sample; DNA was isolated by RNase treatment of the nucleic acid sample. An equal volume of each was analyzed on the Agilent 2100 bioanalyzer.
Get More Data from Your FFPE Samples
Rick Conrad, Tim Barta, Emily Zeringer • Ambion, Inc.
2. Standard embedding protocols require heating the formaldehyde-soaked samples. The temperatures used for embedding in wax cause further molecular reactions, some irreversible.
3. The RNA in tissue blocks degrades during standard storage. The chemistry behind this is unclear.