Authors (including presenting author) :
Lok, J(1), Lo, YF(1), Li TK(1), Fung HY(1), Shek WH(1)
Affiliation :
Department of Pathology, Queen Mary Hospital
Introduction :
Formic acid is currently the decalcifying agent in our laboratory. However, formic acid is known to cause damage to DNA, thus limiting the availability of high-quality DNA for downstream molecular application. The use of a milder decalcifying agent, such as ethylenediaminetetraacetic acid (EDTA), has been advocated by some authors as it has been shown to yield improved DNA from bone marrow tissues.
Objectives :
To determine and compare the effects of formic acid and EDTA on the quantity and quality of DNA extracted from bone tissue.
To determine and compare the duration of the decalcification process by formic acid and EDTA.
Methodology :
Femoral heads of 19 patients were retrospectively selected for study. A 4-mm slice of bone had been previously sectioned and decalcified using 15% formic acid according to protocol. Two 4-mm bone slices were sectioned from the left-over material, fixed in 10% neutral buffered formalin, and decalcified using EDTA. The endpoint of the decalcification was determined based on ‘physical’ and ‘chemical’ methods.
DNA from EDTA-decalcified bone was then extracted, using commercially available DNA isolation kits and compared to DNA extracted from formic acid-decalcified bone that was originally used for diagnosis.
DNA quantity was determined by UV-spectrophotometer. DNA quality was assessed by amplifying DNA fragments of different base pair lengths (incl. 135bp, 315bp, and 480bp).
Result & Outcome :
DNA quality from EDTA-decalcified bone was markedly better. The percentage of EDTA-decalcified bone, yielding 135bp-, 315bp-, and 480bp-long DNA fragments were 69% (N=36), 75% (N=30), and 73% (N=19), respectively. In contrast, 31% (N=16), 25% (N=10), and 27% (N=7) of formic acid-decalcified bone generated 135bp-, 315bp-, and 480bp-long DNA fragments, respectively.
Detecting the endpoint of decalcification whether by the ‘physical’ or ‘chemical’ method resulted in no marked difference in the quality and quantity of DNA.
On average, decalcification by EDTA was 3.2 days vs. 1.5 days for formic acid.
The study showed a trend that bone tissue decalcified by EDTA resulted in an overall improved DNA quality. Such DNA may be better suited for downstream molecular applications. While turn-around time for EDTA decalcification will invariably be prolonged, the improved DNA quality may prove invaluable as molecular diagnostics and therapeutics play an increasingly important role in patient care.
