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 m 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 (see Examine MicroRNA Profiles from Archived FFPE Tissue).
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 80C; the other half was fixed and embedded using a standard hospital protocol. RNA was isolated from one 20 m 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 m 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.
Rick Conrad, Tim Barta, Emily Zeringer Ambion, Inc.
Cat# Product Name Size 1710 RETROscript Kit 40 rxns 1975 RecoverAll Total Nucleic Acid Isolation Kit for FFPE 40 purifications 2054 SuperTaq Plus Polymerase (Cloned) 5 U/l 50 U 2056 SuperTaq Plus Polymerase (Cloned) 5 U/l 250 U