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Recover High Yields of Total Nucleic Acid from Formalin-fixed, Paraffin-embedded (FFPE) Tissue

(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 prim
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