Effect of Different Proteinase K Digest Protocols and Deparaffinization Methods on Yield and Integrity of DNA Extracted From Formalin-fixed, Paraffin-embedded Tissue

Abstract

DNA extracted from formalin-fixed, paraffin-embedded tissue sections is often inadequate for sequencing, due to poor yield or degradation. We optimized the proteinase K digest by testing increased volume of enzyme and increased digest length from the manufacturer's protocol using 54 biospecimens, performing the digest in centrifuge tubes. Doubling the quantity of proteinase K resulted in a median increase in yield of 96%. Applying the optimized proteinase K protocol to sections deparaffinized on microscope slides generated a further increase in yield of 41%, but only at >50,000 epithelial tumor cells/section. DNA yield now correlated with (χ² = 0.84) and could be predicted from the epithelial tumor cell number. DNA integrity was assayed using end point multiplex PCR (amplicons of 100-400 bp visualized on a gel), quantitative PCR (qPCR; Illumina FFPE QC Assay), and nanoelectrophoresis (DNA Integrity Numbers [DINs]). Generally, increases in yield were accompanied by increases in integrity, but sometimes qPCR and DIN results were conflicting. Amplicons of 400 bp were almost universally obtained. The process of optimization enabled us to reduce the percentage of samples that failed published quality control thresholds for determining amenability to whole genome sequencing from 33% to 7%.

Keywords: DNA Integrity Number; Illumina FFPE QC Assay; genomic screen tape; quality control.

Figure 1.

Influence of proteinase K digest on DNA yield. The boxes are the 25th to 75th percentiles, intersected by the median. The whiskers show the 10th and 90th percentiles, and each spot represents one remaining data point. (A) The 40 µl/24 hr digest returned the highest yield. The difference between the three protocols is statistically significant in all pairwise comparisons (p<0.05). (B) Changes in DNA yield, relative to the 20 µl/24 hr proteinase K protocol, generated by increasing the proteinase K digest volume to 40 µl compared with changes induced by increasing the digest length to 72 hr. Tissue blocks tended to have a shared degree of resilience to either change in protocol. Each data point is a tissue block. The white arrows identify the six blocks with the greatest increase in yield when the digest time was lengthened, as discussed in the text. (C) There was a moderate positive correlation between DNA yield and cellularity in the 20 µl/24 hr digest (see text), the magnitude of which increased in proportion to epithelial tumor cellularity when the volume of proteinase K was increased to 40 µl (black circles). However, the magnitude of the change in DNA yield was unrelated to cellularity when the digest length was increased to 72 hr (white circles). Black and white circles that are aligned vertically are the two digests from the same tissue block. Note the logarithmic scale in the x-axis.

Figure 2.

Effect of changing the deparaffinization protocol on DNA yield. (A) Changing the deparaffinization protocol from tubes to slides generated an increase in DNA yield (p<0.001). The parameters of the box plot are as in Fig. 1A. (B) The magnitude of the increase in DNA yield gained when switching to slide deparaffinization was dependent on the cellularity of the sample, but only at >50,000 epithelial tumor cells per section. Note the logarithmic scale in the x-axis. (C) When deparaffinization was performed on slides, DNA yield correlated to the number of tumor cells using the equation √DNA yield (ng DNA/section) = −28.45 + (9.742 × log₍₁₀₎ no. of tumor cells). The linear regression line is shown. (D) Comparison of the DNA yield predicted using the epithelial tumor cell count and the yield assayed by Pico Green spectrofluorometry.

Figure 3.

Effect of different Proteinase K digest protocols and deparaffinization methods on DNA Integrity Number (DIN). (A) When deparaffinization was performed in tubes, the 40 µl/24 hr proteinase K digest had the highest median DIN and a considerably higher 25th percentile DIN than either of the digests using 20 µl proteinase K. When 20 µl proteinase K was used, extending the digest time from 24 to 72 hr had no statistically significant effect. (B) Median DINs and the 75th percentile were both slightly higher when sections were deparaffinized in tubes compared with on microscope slides. Although the difference was statistically significant, it was very small in magnitude. The parameters of the box plot are as in Fig. 1A, and the horizontal lines with start and end dots denote the groups that are statistically significantly different from each other (p<0.05).

Figure 4.

Effect of different proteinase K digest protocols and deparaffinization methods on DNA integrity assayed using the Illumina FFPE QC Assay. (A) The differences between the three proteinase K protocols were not statistically significant. (B) When the 40 µl/24 hr proteinase K digest was performed, switching to deparaffinization on slides lowered the ∆Cq values. Lower ∆Cq values denote improved DNA integrity. The parameters of the box plot are as in Fig. 1A, and the horizontal lines with start and end dots denote the groups that are statistically significantly different from each other (p=0.01).