Fig 1.
DNA quality and quantity of various clinical diagnostic FFPE tissues.
DNA was extracted from 27 FFPE samples of normal breast, normal liver and tumorous tongue and then qualified and quantified. The samples were divided into two groups to test the formalin concentration and subdivided for the number of fixation days. Six tongue tissues were fixed with 10% neutral buffered formalin. The four samples with black dots were fixed at a temperature higher than 25°C. (A) The DNA quality was determined by qPCR of human GAPDH (93 bp), and the results are expressed as the ratio to fresh-frozen DNA. (B) The DNA yield was determined using an UV spectrophotometer, and the results are expressed as DNA quantity per cm2 of tissue area with 10 μm thickness.
Fig 2.
FFPE DNA quality is dependent on the formalin fixation duration.
Three sets of 5 matched samples containing fresh-frozen tissue DNA and 4 FFPE (1-, 2-, 3- and 4-day fixation) DNA samples were prepared from 3 rat liver specimens (r1, r2, r3) and examined for DNA quality. The data are shown as averages (circles) with standard deviations (bars).
Fig 3.
Agarose gel electrophoresis of rat liver DNA.
(A) Agarose gel electrophoresis (0.8%) of 400 ng of purified DNA. DNA on the gel were stained with GelRed which is sensitive to double-stranded DNA. The qPCR ratio to matched frozen tissue DNA is shown at the bottom of each lane. Fro, frozen tissue DNA; Fix., fixation for 1 day (1 d): lanes 2 and 3 were different tissue samples with 1 day fixation, 4 days (4 d) and 5 days (5 d); Heat, heat treatment of lysate (+) or not (-) at 95°C for 30 minutes; M, molecular weight marker; qPCR, qPCR ratio to frozen tissue DNA. (B) Agarose gel electrophoresis (2%) of PCR products with target sizes of 301 bp (a) and 952 bp (b). The rat Tp53 gene was amplified from the indicated DNA, lanes 1 to 8 in (A).
Fig 4.
Effects of heat treatment of FFPE lysates on DNA quality and quantity.
(A) Rat liver FFPE specimens: 6 matched frozen and FFPE tissues (r1 to r3 fixed for 4 days and r4 to r6 fixed for 5 days shown in S1 Fig) were used, and the FFPE tissue lysates were treated (+) or not (-) at 95°C for 30 minutes before purification. DNA quality and quantity were determined as shown in Fig 1. (B) Human normal liver FFPE specimens: 4 matched frozen and FFPE tissues (h16, h18, h19 and h22) were used, and the FFPE tissue lysates were heat treated and examined for DNA quality (left) and quantity (right), as described in (A). **, p<0.01; *, p<0.05 by the paired t-test.
Fig 5.
FF DNA quality and quantity dependent on the heat treatment.
Three sets of 5 matched samples containing fresh-frozen and FF (1-, 2-, 3- and 4-day fixation) tissues were prepared from 3 rat liver specimens (r1, r2, r3). FF DNA was extracted from the lysates with (+) or without (-) heat treatment and examined for DNA quality (left) and DNA quantity (right).
Fig 6.
DNA quality and quantity during the course of IHC staining of FFPE thin sections.
(A) The DNA qPCR ratio and relative DNA yield were expressed using FFPE thin sections before the start of IHC used as the control. The IHC procedure was divided into 6 steps. Four FFPE samples (h16 to h19) were used for human hepatocyte marker immunostaining. The qPCR value and DNA yield relative to the matched control were calculated, and the mean values of 4 samples were compared among the 6 steps. Control DNA was prepared directly from matched FFPE thin sections according to our modified RecoverAll protocol with additional heat treatment. (B) Agarose gel (0.8%) electrophoresis of 3 representative sets (h16, h17 and h19) of matched DNA samples (500 ng/lane). Fr, frozen tissue DNA; a, b, c, g, DNAs as shown in (A).
Fig 7.
Venn diagram of variant candidates found in 4-pair analyses of FFPE and frozen tissue DNA.
Variants identified in 4 matched FFPE-frozen pairs of normal liver tissues, h16, h17, h18 and h19. Fourteen variants were identified by pair analysis using Ion Reporter version 5.0 with the following filters: coverage more than 100, allele frequency more than 0.05, frozen allele coverage = 0. ERCC2 was a common variant among two samples, h16 and h17.
Fig 8.
Sanger sequencing of FGFR3, CHEK2, and MAGI1.
Variant calls at high frequency were invalidated by Sanger sequencing of amplified products from matched fresh-frozen DNA (upper panel) and FFPE DNA (lower panel). Arrows indicate the positions of a deletion (FGFR3), an SNV (CHEK2) and an insertion (MAGI1), and the variant sequences are shown below the reference sequences.
Table 1.
Coverage depth of amplicon sequencing of the comprehensive Cancer Panel.
Table 2.
Nineteen variant candidates.
Table 3.
Invalidation of the variant candidates by allele-specific qPCR.