The Biospecimen Research Database

The purpose of this study was to determine the extent that DNA and RNA quantity and quality (integrity) are affected by a delay to fixation and the duration of formalin fixation in FFPE tumor specimens. The BPV Program's Scientific Steering Committee identified delay to fixation (DTF) and time in fixative (TIF) as pre-analytical factors requiring additional study. To address this, primary tumor specimens were collected from patients diagnosed with renal cell carcinoma (kidney), colorectal adenocarcinoma (colon), and ovarian/fallopian tube carcinoma (ovary) at four different medical centers. The effects of DTF were investigated in kidney, ovary, and colon tumor aliquots subjected to a room temperature delay of 1, 2, 3, or 12 h in a humidified chamber before fixation in 10% neutral buffered formalin (NBF) for 10-12 h. Effects of TIF were investigated in kidney and ovary tumor aliquots fixed in 10% NBF at room temperature for 6, 12, 23 of 72 h within 1 h of collection. Two additional tumor aliquots were collected from each patient and preserved within 1 hour of collection and either snap-frozen in liquid nitrogen (LN2) and stored in LN2 vapor for use as a gold standard, or fixed in 10% NBF at room temperature for 23 h to verify that cases met histopathology eligibility criteria (>50% tumor content by surface area, <20% necrosis); although eligibility was ultimately determined for each FFPE block.  RNA and DNA were extracted from FFPE sections using the QIASymphony RNA kit and QIASymphony DNA mini kit, respectively, albeit different protocols were used for snap-frozen and FFPE specimens. Nucleic acid concentration, purity, and potential contamination were assessed by Nanodrop spectrophotometry. DNA and RNA quantity was determined by fluorometry with corresponding Qubit kits. DNA quality (integrity) was determined by the qPCR-based KAPA Human Genomic DNA Quantification and QC kit and reported as a Q-ratio, a ratio of medium (129 bp) or long (305 bp) length amplicons relative to a short (41 bp) amplicon for a conserved single-copy gene. RNA quality (integrity) was assessed by RIN and DV200 scores that were generated with an Agilent bioanalyzer. RNA quality (integrity) was also assessed by a qRT-PCR-based assay and reported as a Q-score, a ratio of medium (165 bp) to short (80 bp) amplicons of GAPDH or PGK1. Although the number of specimens analyzed depended on the experimental timepoint and assay, 17 to 29 kidney, 4-10 ovary, and 9-13 colon tumor specimens were analyzed. All experimental timepoints were randomly assigned and researchers were blind to these assignments during analysis.