TO THE EDITOR
Extraction of DNA from bone marrow slides for molecular biology studies is a standard technique for which multiple commercial DNA extraction kits are available. Slides of bone marrow and other tissues may also be used as a DNA source for molecular epidemiological studies.1 Adequate amounts of DNA may be obtained from both stained and unstained slides,2 DNA may be obtained from specimens archived for decades,3 and different phenol-chloroform extraction procedures give varied DNA yields.4 Since phenol-chloroform reactions are time-consuming and expose laboratory personnel to toxic reagents, research laboratories most often use commercial kits for DNA extraction. Verhagen et al5 have shown that DNA yields adequate for Southern blotting may be obtained from bone marrow specimens with the Qiagen column, a common commercial DNA extraction kit.
Commercial DNA extraction kits involve various techniques for DNA purification. All kits employ cell lysis and protein denaturations steps to obtain DNA for purification. Some kits use a silica-gel based column to bind DNA, followed by serial washes to remove non-DNA cell contents and DNA elution in a low salt buffer (Qiagen). Other kits use a 'salting out' procedure with alcohol extraction (Puregene) to isolate and purify DNA.
Despite the widespread use of commercial kits, no formal comparison of the yields and purity of DNA extracted by these kits have been reported. In general, research laboratories using commercial DNA extraction kits obtain adequate DNA yields from samples with sufficient numbers of nucleated cells. However, the archived bone marrow slides often used for molecular epidemiology studies generally contain small numbers of nucleated cells with fragmented DNA. Little information is known about DNA yields obtained from such samples with commercial DNA extraction kits.
After considering multiple extraction kits, we chose to evaluate the Qiagen DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) and the Puregene DNA Tissue DNA Extraction Kit (Gentra Systems, Minneapolis, MN, USA) based on their widespread use, different methodologies, and familiarity of the company's technical support staff with bone marrow aspirate slide specimens. The extraction procedures described below were modified to give maximum DNA yields, in consultation with the technical support staff from each company. Modified extraction protocols were compared to unmodified extraction protocols and consistently gave higher DNA yields (data not shown).
We compared these kits on fresh bone marrow specimens, stained and unstained bone marrow slides made from fresh specimens, and archival samples. Newly collected discarded bone marrow aspirate samples were obtained from the Outpatient Hematology laboratory at the Children's Hospital of Philadelphia (CHOP). Samples were chosen randomly from normal, chemotherapy exposed, and leukemic bone marrow samples. This range of sample types was chosen to mimic the range of bone marrow samples typically available from slide specimens. No identifying information or patient data were linked to the discarded sample. The number of cells per  l was estimated with a hemocytomer. Five l of bone marrow was then smeared on a glass slide using standard laboratory techniques. Multiple slides were made per patient sample and the slides were allowed to air dry. At least two slides per sample were stained with Wright-Giemsa stain. Ten archived bone marrow specimens made prior to 1980 were selected at random from the slide archive. Cover slips were removed by soaking the slides in xylene for 48 h. Each coverslip was then rinsed in ethanol to remove any remaining xylene and allowed to air dry prior to DNA extraction as described below. All personal identifiers were removed from the specimen prior to DNA extraction.
Fresh marrow was prepared for DNA extraction by diluting 5 l of marrow in either 195 l of PBS or 195 l of Puregene Cell Lysis Solution. Marrow was removed from slides and cover slips by wetting the marrow specimen with approximately 10 l of either phosphate-buffered saline (PBS) or the Purgene proprietary cell lysis solution. The bone marrow was then scraped off the slide or cover slip into an Eppendorf tube. A fresh razor blade was used for each extraction and the laboratory bench was cleaned with 70% ethanol between extractions to minimize cross-contamination. Gloves were changed between each patient sample. All extractions were performed in duplicate.
The manufacturer's recommended protocol for the Qiagen extraction was modified by prolonging the AW1 and AW2 wash times to 5 min. A second spin after the AW2 wash was added. The proprietary Elution Buffer AE was heated to 65°C prior to elution and the eluate was applied twice to the spin column. The manufacturer's recommended protocol for the Puregene extraction was modified by incubation of the Proteinase K step overnight, cooling the sample at 4°C for 5 min prior to protein precipitation, and cooling the sample to -20°C for several hours during the isopropanol and ethanol precipitation steps. All samples were eluted in 50 l of proprietary elution buffer and stored at 4°C prior to DNA quantification and PCR use.
DNA yields of all extractions were estimated by DNA spectrophotometry (GeneQuant and GeneQuant Pro, Amersham Pharmacia, Cambridge, UK) according to the manufacturer's instructions. PCR amplification efficiency was semi-quantitatively assessed by amplification of a 280 base pair  -globin product. PCR yields were estimated by comparison of band intensity to a DNA Mass Ladder (Roche Molecular Biochemicals, Indianapolis, IN, USA) on a 2% agarose gel stained with ethidium bromide by blinded reviewers who had not participated in DNA extraction, DNA quantification, or PCR preparation. DNA yields, DNA 260/280 ratios, and PCR yields of identical patient extraction types were averaged prior to analysis and were compared as paired data between kits with the Wilcoxon sign rank test.
The number of nucleated cells in bone marrow varied widely, from 12 500 to 421 500 cells per 5 l with a mean of 233 413 (median 181 000, s.d. 179 013, Table 1). The amount of genomic DNA obtained from each kit is summarized in Table 1. Notably, the Puregene kit gave consistently higher DNA yields than the Qiagen kit. DNA obtained from both kits had similar 260/280 ratios. The average PCR yield from fresh marrow specimens was 85 ng for the Puregene kit and 48 ng for the Qiagen kit (Wilcoxon sign rank test z = 2.812, P = 0.005). The mean PCR yield for unstained slides extracted with the Puregene Kit was 76 ng while the average PCR yield for the Qiagen kit was 120 ng (Wilcoxon sign rank test z = 2.041, P = 0.041). The average PCR yield from stained slides was 82 ng for the Puregene kit and 51 ng for the Qiagen kit (Wilcoxon sign rank test z = 2.197, P = 0.028). The average PCR yield from archived cover slips was 57 ng by the Puregene kit and 46 ng by the Qiagen kit (Wilcoxon sign rank test z = 1.864, P = 0.08).
We conclude that while adequate quality and quantity of DNA can be obtained from small volumes of bone marrow samples and slide cover slips, yields may differ between commercial DNA extraction kits. The Puregene kit in general gave higher DNA and PCR yields than the Qiagen kit. The increased yields in the Puregene kit may be due to the overnight digestion with Proteinase K, which would increase the amount of cell lysis and DNA available for recovery. Interestingly, other investigators have noted increased DNA yields with Proteinase K incubation for 48 to 72 h (unpublished data). Alternatively, incomplete initial DNA binding to the Qiagen silica-based DNA capture column may result in lower amounts of DNA for subsequent recovery in the elution buffer.
Both kits had significant variability in their DNA yields. This variability is due in part to the substantial differences in cell number between bone marrow specimens. DNA yields from the Puregene kit appear to have greater standard deviations than DNA yields from the Qiagen kit. This difference in DNA yield variability may be due to differences in the extraction procedures. The Puregene kit requires DNA precipitation and serial washes that may have relatively higher chances for DNA loss.
In order to obtain maximal DNA and PCR yields, several modifications to the standard DNA extraction procedure was made to both kits. The Puregene extraction was modified by extending the Proteinase digestion overnight, as well as holding the isopropanol and ethanol washes at 20°C for at least 1 h and 30 min, respectively. These changes lengthened the time necessary for each extraction. The Qiagen extraction was modified by adding an additional 5 min spin after the AW2 wash. The second spin removed any remaining alcohol that might inhibit subsequent PCR assays. This change did not significantly change the time necessary for DNA extraction. While both extraction protocols are relatively simple, the Puregene protocol was more labor intensive and may have a higher chance of contamination/DNA loss, as the isopropanol and ethanol washes both require decanting of the washing solution. These differences may make the Puregene kit less amenable to a high throughput DNA extraction process.
In summary, both the Puregene and Qiagen kits yield DNA of adequate quantity and purity of PCR-based genotyping. If maximal DNA yields are required, the Puregene kit may provide higher DNA yields. However, the Qiagen extraction requires less time and is technically easier. Thus, if multiple bone marrow aliquots or slides are available on a subject, then the Qiagen kit may provide a faster and more uniform DNA extraction.
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