A simple and cost-effective method of DNA extraction from small formalin-fixed paraffin-embedded tissue for molecular oncologic testing

Abstract

BackgroundExtraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. As molecular oncology testing becomes more important for prognostic and therapeutic decision making and tissue specimens become smaller due to earlier detection of suspicious lesions and the use of fine needle aspiration methods for tissue collection, it becomes more challenging for the typical molecular pathology laboratory to obtain reliable test results. We developed a DNA extraction method to obtain sufficient quantity and high quality genomic DNA from limited FFPE tissue for molecular oncology testing using a combination of H&E stained slides, a matrix capture method and the Qiagen DNA column.MethodsThree DNA extraction methods were compared: our standard procedure of manually scraping tissue from unstained slides followed by DNA extraction using the QIAamp FFPE column (Qiagen, Valencia, CA), a glue capture method (Pinpoint Solution, Zymo Research Corp, Inc) on H&E stained slides followed by DNA extraction using either the QIAamp column or the column included with the Pinpoint kit (Zymo Research). The DNA extraction protocol was optimized. Statistical analysis was performed using the paired two-sample student’s t-test.ResultsThe combination of the matrix capture method with the QIAamp column gave an equivalent amount of DNA as our standard extraction method using the unstained slides and a 4.6-fold higher DNA yield than using the Zymo column included in the Pinpoint Slide Solution kit. Several molecular tests were performed and DNA purified using the new method gave the same results as for the previous methods.ConclusionsUsing H&E stained slides allows visual confirmation of tumor cells during microdissection. The Pinpoint solution made removal of specific tissue from the slides easier and reduced the risk of contamination and tissue loss. This DNA extraction method is simple, cost-effective, and blends with our current workflow requiring no additional equipment.

Rapid Differentiation of Filariae in Unstained and Stained Paraffin-Embedded Sections by a High-Resolution Melting Analysis PCR Assay

“…Thus, a Pinpoint solution can be used to minimize potential contamination from tissue flakes in the work area (Snow et al 2014). In the present study, we prevented flaking by adding lysis buffer directly onto the section prior to scraping.…”

An Effective and Viable DNA Extraction Protocol for FFPE Tissues and its Effect on Downstream Molecular Application

“…This enzyme acts on proteins by cleaving the peptide bonds adjacent to the carboxyl group of hydrophobic amino acid residues (aliphatic and aromatic). The optimal temperature for proteinase K is known to be between 50-60°C and the specific activity of proteinase K is enhanced by increasing the temperature from 52°C to 56°C (Snow et al, 2014). This will unfold the proteins, exposing the hydrophobic amino acid residues that would be normally encrypted inside the hydrophobic core of the protein making it easier for enzyme action.…”

“…Even though the normal colonic mucosa archival tissue is a smaller biopsy compared to colorectal cancer archival tissue we were able to isolate good quality and quantity of DNA. Snow et al (2014) have reported that extraction of DNA from cellular tumors with a solid growth pattern and less contaminating nonneoplastic tissue is comparatively easy. In contrast to small biopsies which usually contain minute nests of tumor cells surrounded by non-tumor tissue creating a challenge in obtaining adequate quantity of DNA material (Snow et al, 2014).…”

“…Snow et al (2014) have reported that extraction of DNA from cellular tumors with a solid growth pattern and less contaminating nonneoplastic tissue is comparatively easy. In contrast to small biopsies which usually contain minute nests of tumor cells surrounded by non-tumor tissue creating a challenge in obtaining adequate quantity of DNA material (Snow et al, 2014). Therefore our newly modified DNA extraction method enables us to acquire adequate quantity of DNA from diminutive foci of the tumor and can be utilized for both cancer tissues as well as non neoplastic tissues.…”

Utility of different massive parallel sequencing platforms for mutation profiling in clinical samples and identification of pitfalls using FFPE tissue

“…Additionally, the preanalytical steps are important to consider: the manual macrodissection of selected tumour areas has become a standard procedure in molecular pathology and is a powerful tool to reduce false negative results resulting from wild-type contamination ( 10 ). Selecting the right tumour area influences not only the result of the analysis, but also the allele frequency, the value of which is pivotal when reporting diagnostic findings ( 11 ).…”