EPICENTRE Biotechnologies
726 Post Rd.
Madison, WI, 53713
Website: http://www.epibio.com
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Obtain PCR-Ready DNA From FFPE Tissue In 30 Minutes
by Bruce W. Jarvis and Judith E. Meis
Introduction
Figure 1. End-point PCR amplification
of DNA extracted from FFPE mouse embryos. Lane 1, 100 bp ladder; Lane 2,
0.5 hr extraction; Lane 3, 1 hr; Lane 4, 3 hr; and Lane 5, no template control
PCR. The reaction used EPICENTRE Biotechnologies FailSafe PCR System with
PreMix D, and b-actin primers. The primer sequences were, 5’ primer:
5’-TCA TGA AGT GTG ACG TTG ACA TCC GT and 3’ primer: 5’-CTT
AGA AGC ATT TGC GGT GCA CGA TG. Cycling conditions were: 95 C (2 min) and
35 cycles of 95 C (30 sec), 55 C (30 sec), 74 C (30 sec). The amplicon was
285 bp in length. |
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 1918.1
This report provides detail on the use of QuickExtract DNA Extraction Solution (Epicentre Biotechnologies, Madison, WI), which facilitates the rapid extraction of PCR-ready DNA from formalin-fixed, paraffin-embedded 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30 μl aliquots were withdrawn from the solution — the amount of DNA extracted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.
Incubation Time In Quick Extract |
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DNA Extraction Solution (hours) |
0.5 |
1.0 |
3.0 |
Total Yield of DNA (ng) |
ND* |
30 |
90 |
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 the 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 solution.
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Figure 2. qPCR of DNA extracted from FFPE tissues using QuickExtract
DNA Extraction Solution. Duplicate 25 ml qPCR reactions were set up containing: 12.5
ml TAQurate GREEN Real-Time PCR MasterMix, 10 pmoles of forward and reverse
primers for cyclophilin A, 1 U TAQurate Real-Time PCR Enzyme Blend, and
1 ml of appropriate DNA. Cycling conditions were: 95 C (2 min), and 40
cycles of 95 C (20 sec), 57 C (20 sec), and 72 C (30 sec). A. Amplification
plot. The DNA template for PCR was extracted from FFPE tissue 0.5 hours
(green traces), or 3 hours (red traces) before PCR amplification in duplicate.
No DNA template control, and positive controls performed as expected,
data not shown. B. Melt curve analysis of the amplicons of panel A. No
DNA template negative control shown as black trace.
Green = 0.5 hours QE/ FFPEsample
Red = 3.0 hours QE/
FFPE sample
Black = negative control
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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; Sigma 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.
About the authors
Bruce W. Jarvis and Judith E. Meis are with EPICENTRE Biotechnologies in Madison, Wisconsin.
References
1. Reid, A.H. et al. Proc. Natl. Acad. Sci. 97(12):6785 (2000).
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