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.
Highlights
Isolating genomic DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing [1,2,3,4,5,6]
The first group used the standard method in the laboratory (U-SQ), in which unstained FFPE tissue was scraped with a razor blade followed by extraction with the QIAamp kit
The second and third groups employed deparaffinization and hematoxylin and eosin (H&E) staining of the slides followed by tissue harvest using the Pinpoint reagent
Summary
Extraction of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing. 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. Isolating genomic DNA (gDNA) from formalin-fixed, paraffin-embedded (FFPE) tissue is a critical step in molecular oncologic testing [1,2,3,4,5,6]. Laser capture microdissection (LCM) [12,13] is an alternative; but it requires special equipment and special training for the medical technologists It is cost and space prohibitive for many laboratories to invest in an LCM system
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