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Conventional histopathology techniques include the microscopic observation of thin sections of preserved tissues that are stained to reveal anatomical structures. A commonly used method for tissue preservation has been formalin-fixation followed by paraffin-embedding (FFPE). In recent years the desire for molecular information from these preserved, archival tissues has emerged. One of the most notable successful examples of nucleic acid extraction from such an archived FFPE tissue was the retrieval of influenza viral RNA sequence data from the FFPE lung tissue of soldiers who died in 1918.
QuickExtract™ DNA Extraction Solution enables the rapid extraction of PCR-ready DNA from formalin-fixed, paraffinembedded tissues. The DNA can be used in end-point or real-time PCR (qPCR).
Methods
FFPE tissue from a slide (2 months old) of thin sections (0.5 micron) of three mouse embryos (at day 12 of gestation; courtesy of Dr. Igor Prudovsky, Maine Medical Center Research Institute) was scraped with a flamed scalpel blade into 100 μl of QuickExtract Solution and heated at 65°C. At 0.5, 1, and 3 hour time intervals, 30 μl aliquots were withdrawn from the solution—the amount of DNA extracted was quantified by fluorescence with Hoechst dye 33258. The DNA was also amplified by end-point PCR with β-actin primers, and by qPCR with SYBR® Green I dye using beta-2-microglobulin primers.
Results
Table 1 shows the data for the amounts of DNA detected at each time point. As one might expect, the average amount of DNA extracted increased with the length of time of incubation in QuickExtract DNA Extraction Solution.
In FIG 1 it is also possible to spot the trend of increasing amounts of amplicon produced by end-point PCR with increasing length of incubation in QuickExtract Solution.
FIG 2 displays the results from qPCR analysis of cyclophilin A (peptidylprolyl isomerase A) from DNA extracted at varying times from a mixture of FFPE human tissues (at least 3.5 years old; H-2286) scraped from a slide as described above. Cyclophilin A was readily amplified from all samples by qPCR with SYBR Green I dye. As predicted, the CT (threshold cycle) obtained from the three hour sample was significantly lower (indicating greater abundance) than the CT of the 0.5 hour extracted DNA sample. However, there was clearly DNA present after the 0.5 hour digestion.
Conclusion
QuickExtract provides a rapid, simple method for obtaining PCR-ready DNA from archived FFPE tissues in 30 minutes.
Reference
1. Reid, A.H. et al., (2000) Proc.Natl.Acad.Sci. 97(12), 6785.
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