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

Author(s): Frazer Z, Yoo C, Sroya M, Bellora C, DeWitt BL, Sanchez I, Thomas GA, Mathieson W

Publication: J Histochem Cytochem, 2020, Vol. 68, Page 171-184

PubMed ID: 32043912 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to compare the yield, purity and integrity of DNA isolated from FFPE tissue sections using three different proteinase K digestion protocols following deparaffinization on slides or in tubes.

Conclusion of Paper

The median DNA yield was higher when the proteinase K was refreshed after 6 h compared to digestion for 24 h or 72 h without refreshment. DNA yield was modestly correlated with number of epithelial tumor cells per section, but the correlation was slightly stronger when proteinase k was replenished compared to when it was not. Replenishment of proteinase K resulted in a nonsignificantly higher percentage of dsDNA and a significantly higher mean DNA integrity numbers (DIN). DNA purity (absorbance at 260/230 nm and 260/280 nm), 𝛥Cq in the Illumina QC assay, and amplification products in the multiplex PCR assay did not differ among the digestion protocols evaluated. The authors report that deparaffinization method had no affect on DNA yield or integrity in the tissue types examined and report no correlation between storage duration and DNA yield.

However, specimens with >50,000 epithelial tumor cells/section had a higher median DNA yield when deparaffinization occurred on the slide rather than in a tube; DNA yield also increased with cellularity in these specimens. FFPE sections deparaffinized on slides rather than in tubes had a nonsignificantly higher percentage dsDNA; and significantly higher median 𝛥Cq in the Illumina FFPE QC assay and a significantly higher percentage of specimens that meet the threshold of 𝛥Cq. The 𝛥Cq was modestly correlated to the number of epithelial tumor cells. Specimens with the greatest cellularity demonstrated the largest increase in DNA yield with proteinase k replenishment or deparaffinization on the slide and had a significantly higher 𝛥Cq. DNA concentration was very strongly correlated between the two fluorometric based kits examined.

Studies

Study Purpose

The purpose of this study was to compare the yield, purity and integrity of DNA isolated from FFPE tissue sections using three different proteinase K digestion protocols. A total of 54 tumor blocks containing breast (17), kidney (5), bladder (1), prostate (1), carcinosarcoma (2), ovary (15), uterus (3), endometrium (6), fallopian tube (1), omentum (1), colon (1), and adenoid carcinoma (1) tumors were obtained from three different hospitals. All specimens were fixed for 24 h in 10% neutral buffered formalin and processed into FFPE blocks using the Tissue-Tek Vacuum Infiltration Processor 5 and the manufacturer-specified protocol. After embedding, blocks were stored in the dark at room temperature for 34-313 days before sectioning. The number of tumor cells was manually counted in a single 4 µm-thick H&E stained section. A scroll of 10 4 µm-thick sections were placed in each tube, deparaffinized in Histoclear for 10 sec and washed in ethanol. DNA was then extracted using the QIAamp DNA FFPE Tissue Kit with one of three different proteinase K digestions (all at 56°C): 20 µL proteinase K for 24 h, 20 µL proteinase K for 5 h and then another 20 µL proteinase K for remaining 19 h, 20µL proteinase K for 72 h. The optional RNAse step was included for all extractions. DNA concentration and purity were evaluated by spectrophotometer; the concentration of double-stranded DNA was evaluated using the QuBit dsDNA broad range assay kit and the Quant-iT dsDNA assay. DNA integrity was assessed by TapeStation, the real-time PCR-based Illumina FFPE QC Assay, and by conventional multiplex PCR of GAPDH.

Summary of Findings:

DNA yield was quantifiable in 100% of FFPE specimens when proteinase K was refreshed after 6 h, in 94% (51/54) of specimens that were digested for 72 h without replenishment, and 88% (48/54) of specimens that were digested for 24 h without replenishment. Median DNA yield was higher when proteinase K was refreshed after 6 h (210 ng/ section) then when the digestion was for 24 h or 72 h without replenishment (107 ng/section and 112 ng/section, respectively; p<0.05, both). While DNA yield was higher in 65% of specimens when proteinase K was refreshed compared to when it was not, the slight increase in median DNA yield when digestion was extended from 24 to 72 h was only observed in 41% of the blocks evaluated (particularly those with low yields). The increase in DNA yield after proteinase K replenishment was modestly correlated with the increase when digestion was extended (χ²=0.68). Importantly 39% of specimens had higher DNA yields both when proteinase K was replenished and when digestion was extended, but the median DNA yield from these specimens was much higher in those blocks when the Proteinase K was replenished rather than the duration extended (192 ng/section versus 57 ng/section, P=0.009). DNA yield was modestly correlated with number of epithelial tumor cells per section, but the correlation was slightly stronger when proteinase k was replenished (χ²=0.57, P<0.001) compared to when it was not (χ²=0.43, P=0.004 for 24 h and χ²=0.42, P=0.005 for 72 h). The increase in DNA yield observed with proteinase K replenishment was greatest in specimens with the highest number of cells per section. DNA purity (absorbance at 260/230 nm and 260/280 nm) was not affected by the digestion protocols evaluated. The percentage of dsDNA was non-significantly higher when proteinase K was replenished (51.9% versus 36.8% for 24 h and 44.0% for 72 h). Median DINs were also non-significantly higher in proteinase K replenished specimens (6.1 versus 5.7 for 24 h and 5.6 for 72 h, P<0.05, both). The 𝛥Cq values determined by the Illumina QC assay were comparable among the proteinase K digestion protocols evaluated., but the percentage of specimens that exceeded the threshold of 𝛥Cq <2 was higher when proteinase K was replenished (18% versus 7% for 24 h and 4% for 72 h). However, the authors note that this can be deceiving; if specimens that did not yield quantifiable DNA were taken into account, then the failure would be 18% for a 24 h digestion and 10% for a 72 h digestion without proteinase K replenishment. Amplification of all four products was possible regardless of deparaffinization method (in tube versus on the slide) in all but one specimen, where amplification was only successful for fragments ≤200 bp regardless of extraction method. DNA concentration was very strongly correlated between the two fluorometric based kits evaluated (χ²=0.99, P<0.001). The authors report comparable results among the tissue types examined with regard to effects of deparaffinization method on DNA yield or integrity, and observed no correlation between storage duration and DNA yield.

Biospecimens

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
DNA	Automated electrophoresis/Bioanalyzer
DNA	Fluorometry
DNA	Spectrophotometry
DNA	Real-time qPCR
DNA	PCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Protein digestion	20 µL proteinase K for 24 h 20 µL proteinase K for 5 h and then another 20 µL proteinase K for remaining 19 h 20 µL proteinase K for 72 h
Storage	Storage duration	34-313 days
Fluorometry Specific	Technology platform	Tech QuBit dsDNA broad range assay kit Quant-iT dsDNA assay
PCR Specific	Targeted nucleic acid	GAPDH

Study Purpose

The purpose of this study was to compare the yield, purity, and integrity of DNA extracted from FFPE sections that were deparaffinized on a slide or in a tube. A total of 54 tumor blocks containing breast (17), kidney (5), bladder (1), prostate (1), carcinosarcoma (2), ovary (15), uterus (3), endometrium (6), fallopian tube (1), omentum (1), colon (1), and adenoid carcinoma (1) tumors were obtained from three different hospitals. All specimens were fixed for 24 h in 10% neutral buffered formalin and processed into FFPE blocks using the Tissue-Tek Vacuum Infiltration Processor 5 and the manufacturer-specified protocol. After embedding, blocks were stored in the dark at room temperature for 34-313 days before sectioning. The number of tumor cells was manually counted in a single 4 µm-thick H&E stained section. A scroll of 10 4 µm-thick sections were placed in each tube, deparaffinized in Histoclear for 10 sec and washed in ethanol or deparaffinized on slides with Histoclear and then scraped into tubes. DNA was then extracted using the QIAamp DNA FFPE Tissue Kit with proteinase K digestion at 56°C for 5 h replenishment of the proteinase K and digestion for 19 h. DNA concentration and purity were evaluated by spectrophotometer; the concentration of double-stranded DNA was evaluated using the QuBit dsDNA broad range assay kit and the Quant-iT dsDNA assay. DNA integrity was assessed by TapeStation, the real-time PCR-based Illumina FFPE QC Assay, and by conventional multiplex PCR of GAPDH.

Summary of Findings:

DNA yields were greater when FFPE sections were deparaffinized on the slide rather than as a scroll in a tube (297 ng/section versus 210 ng/section, P<0.001). The increase in DNA yield was only observed in specimens with >50,000 epithelial tumor cells/section, and DNA yield also increased with cellularity in these specimens. The correlation between DNA yield and number of epithelial tumor cells was stronger when FFPE sections were deparaffinized on slides rather than in tubes (χ²=0.84 and χ²=0.57, P<0.001). The percentage of dsDNA was non-significantly higher, but the median DIN was non-significantly lower when sections were deparaffinized on slides rather than tubes (52.3% versus 51.9% and 6.0 versus 6.1). The median 𝛥Cq in the Illumina FFPE QC assay was significantly higher for specimens deparaffinized in tubes rather than on slides (0.67 versus 0.26, P=0.01), and the percentage of specimens that met the threshold of 𝛥Cq<2.0 increased from 83% to 91%. The 𝛥Cq was modestly correlated with the number of epithelial tumor cells (χ²=0.42, P=0.006 for tube deparaffinized and χ²=0.43, P=0.001 for slide deparaffinized), and was significantly higher in sections with the greatest cellularity compared to those with the least (1.46 versus -0.21, P<0.001).

Biospecimens

Preservative Types

Formalin

Diagnoses:

Neoplastic - Carcinoma

Platform:

Analyte	Technology Platform
DNA	Automated electrophoresis/Bioanalyzer
DNA	Fluorometry
DNA	Spectrophotometry
DNA	PCR
DNA	Real-time qPCR

Pre-analytical Factors:

Classification	Pre-analytical Factor	Value(s)
Analyte Extraction and Purification	Deparaffinization	On slide In tube

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