Figure 1.
Degradation of DNA from FFPE Tissues and its Effect on PCR Amplification of Amplicons with Different Sizes.
Analysis of DNA integrity of genomic and bisulfite-converted DNA from unfixed and FFPE tissues by means of (A) agarose gel electrophoresis and (B) end-point PCR using PCR fragments of different sizes within the PITX2 gene locus. (C) qPCR results applying increasing amounts (2.5–3,840 ng) of genomic template DNA from unfixed and (D) from FFPE tissue. Shown are the mean values (± standard deviations) from triplicate measurements. Each PCR was performed with 1 U Taq polymerase. DNA from unfixed specimens is considered high molecular weight (HMW) DNA.
Figure 2.
Inhibitory Effect of Template DNA from FFPE Tissues on PCR Performance.
qPCR with 1 µg of genomic HMW template DNA and increasing amounts (60-1,440 ng) of spiked genomic template DNA from FFPE tissue Genomic DNA from FFPE tissue was treated beforehand with active DNase I (+) and heat-inactivated DNase I (-), respectively. qPCR was performed using a 150-bp fragment and 1 U Taq polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.
Figure 3.
Reduction of PCR Inhibition by Increased Amounts of Taq Polymerase.
qPCR with HMW and template DNA from FFPE tissue using different amounts of Taq polymerase. PCR-amplification of increasing amounts of genomic HMW template DNA (2.5-3,840 ng) using (A) 2 U Taq, (B) 4 U Taq and PCR-amplification of template DNA from FFPE tissue using (C) 2 U Taq, (D) 4 U Taq. Shown are the mean values (± standard deviations) from triplicate measurements.
Figure 4.
Successful PCR Amplification of Larger Fragments by Overcoming PCR Inhibition.
PCR-amplified DNA fragments of different sizes within the PITX2 gene locus using template DNA from FFPE tissue. The PCR was carried out using 1 µg (upper and middle panel) and 5 ng (lower panel) template DNA in the presence of 1 U and 4 U Taq DNA polymerase, respectively.
Figure 5.
Transferability of the Findings to a qPCR Targeting an Alternative Genomic Locus.
qPCR applying a 200-bp PCR fragment within the ACTB gene locus. Amplification with (A) HMW DNA from unfixed tissue and (B) DNA from FFPE tissue (2.5 ng to 3,840 ng) in the presence of 1 U and 4 U Taq DNA polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.
Figure 6.
PCR Inhibition by Template DNA from FFPE Tissues with Regard to PCR Conditions (dNTP Concentration and Thermal Cycling Profile).
Shown are qPCR results applying a 200-bp fragment within the PITX2 gene locus using template DNA from FFPE tissue (80-1,920 ng) in the presence of 1 U Taq polymerase. (A) dNTP concentrations and (B) annealing and elongation times were varied. Shown are the mean values (± standard deviations) from triplicate measurements.
Figure 7.
Inhibitory Effect of Template DNA from FFPE Tissues on PCR with Pfu Polymerase.
A 75-bp PCR fragment within the PITX2 gene locus was amplified using genomic template DNA from FFPE tissue (placenta) in the presence of 1 U and 2 U Pfu polymerase. Shown are the mean values (± standard deviations) from triplicate measurements.
Figure 8.
PCR Inhibition by Bisulfite-Converted Template DNA from FFPE Tissues.
(A) qPCR–amplification of different amounts (10–3,840 ng) of bisulfite-converted template DNA from FFPE tissue using a 129-bp PCR fragment within the ACTB gene locus. 1 U and 4 U Taq polymerase were used for qPCR. Shown are the mean values (± standard deviations) from triplicate measurements. (B) PCR amplification of the specific PCR product was confirmed by agarose gel electrophoresis using 1 U (upper panel) and 4 U (lower panel) Taq polymerase.