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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
Ambion has developed a new method for isolating nucleic acids from human and mouse FFPE tissue. Up to four 20 µm sections or unsectioned core samples <35 mg can be processed per reaction. Following deparaffinization and a rigorous protease digestion, the sample can be split into separate aliquots for isolation of total RNA (including miRNA) and genomic DNA using an optimized glass-fiber filter purification protocol.
Total RNA Isolation from FFPE Samples
Although the
RecoverAll Kit cannot reverse RNA fragmentation that may have already occurred (see sidebar, Challenges of Molecular Analysis of FFPE Tissues), the protease digestion conditions are designed to release a maximal amount of RNA of all sizes in a significantly shorter period of time (3 hr) compared to currently available methods. Recovery of mRNA has been verified by qRT-PCR (see below), and microRNAs have been documented with both Northern blot and array analysis.
Gene Expression from FFPE Samples
Figure 1 shows the results of several real-time RT-PCR experiments comparing detection of a variety of genes from fixed versus frozen mouse brain samples. The Ct values from an equal mass of the FFPE samples were higher than those from the frozen controls, indicating that other modifications from the fixation process have adversely affected the RNA as a reverse transcription template.
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.
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
Researchers can now recover DNA from FFPE samples just as easily as RNA by simply increasing the digestion time to 2 days. Isolation of DNA includes an RNase treatment to fully eliminate RNA. Figure 2 shows an Agilent® 2100 bioanalyzer scan of total nucleic acids, RNA, and DNA from an FFPE sample processed with the RecoverAll Kit.
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.
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
Many researchers considered their archives of FFPE tissues lost to molecular analysis. Now, with Ambion's RecoverAll Kit, you can isolate the full range of nucleic acids from a single FFPE tissue sample.
Scientific Contributors
Rick Conrad, Tim Barta, Emily Zeringer • Ambion, Inc.
Scientific Contributors
Rick Conrad, Tim Barta, Emily Zeringer • Ambion, Inc.
Challenges of Molecular Analysis of FFPE Tissues1. Formaldehyde is a promiscuous cross-linker. It reacts primarily with proteins, creating a tightly-locked three-dimensional network of proteins, which is also linked to other macromolecules.
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.
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.