Evaluation of DNA extraction from granulocytes discarded in the separation medium after isolation of peripheral blood mononuclear cells and plasma from whole blood
© Murray and Rajeevan; licensee BioMed Central Ltd. 2013
Received: 28 March 2013
Accepted: 29 October 2013
Published: 1 November 2013
Whole blood is generally processed for plasma and peripheral blood mononuclear cells (PBMCs) from granulocytes/erythrocytes using gradient centrifugation of blood with Histopaue-Ficoll. After separation of plasma and PBMCs, the residual erythrocytes/granulocytes, a rich source of DNA, is often discarded along with the separation medium. In order to isolate DNA from the granulocytes, current methods require the removal of the separation medium and subsequent purification of granulocytes. This report provides a method for extracting DNA using the PAXgene Blood DNA kit from granulocytes without purifying them from the separation medium.
Based on 719 erythrocyte/granulocyte samples stored frozen for approximately 10 years in Ficoll-Hypaque separation medium, the mean yield of DNA was 395 μg (median = 281 μg; range = 1.36 to 2077.2 μg), with mean A260/A280 ratio of 1.84 (median = 1.84; range = 1.17 to 2.23). The quality of isolated DNA was sufficient for use as a template for restriction enzyme digestion, real-time PCR, pyrosequencing, and gel based variable number tandem repeats (VNTR) genotyping.
By demonstrating the extraction of substantial amounts of high quality granulocytes DNA without purifying them from the separation medium, this method offers laboratories and biobanks a flexible and cost-effective approach to obtain plasma, PBMCs, and large amounts of DNA from a single blood collection for a variety of molecular genetics/epidemiologic studies.
Centers for Disease Control & Prevention (CDC) Human Subjects Committee approved this study that adhered to the human experimental guidelines of the US Department of Health and Human Services and the Helsinki Declaration. All subjects provided informed consent for the study. Peripheral blood was collected in 8 ml sodium citrate tubes, and PBMCs and plasma fractions were removed using Ficoll . The residual solution containing the Ficoll layer and the erythrocytes/granulocytes was stored in a 50 ml centrifuge tube and frozen at −20°C until DNA extraction (approximately 10 years later). Frozen samples were thawed at 37°C in a water bath and DNA was extracted using the PAXgene Blood DNA Kit (Qiagen, Valencia, CA, USA). The volume of solution containing the separation medium, erythrocytes, and granulocytes was kept constant at 8 ml with addition of phosphate buffered saline solution (pH 7–7.2) as needed. Subsequently, we followed the PAXgene Blood DNA extraction protocol with these samples. DNA concentration and purity were measured using the Nanodrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and the extract was transferred to a 1.5 ml tube and stored at −70°C.
In conclusion, our work demonstrates that DNA can be extracted directly from the residual solution containing the Ficoll layer and erythrocytes/granulocytes left after the isolation of PBMC’s and plasma from whole blood, and that the presence of the separation medium is not a hindrance to extract DNA using the commercially available PAXgene Blood DNA Kit. The quality and quantity of DNA obtained from granulocytes with the separation medium is sufficient enough for a variety of molecular genetics assays. Combining quality and yield, 94.3% (678/719) samples yield >50 μg DNA with an A260/A280 ratio between 1.7 and 2.0. We recommend that genomic DNA from the remaining small percentage of samples (5.7%) with low yield and low A260/A280 ratio may be subjected to whole genome amplification if they need to be recovered in certain situations. Unless purified granulocytes are specifically needed as part of study objective, many laboratories often discard the erythrocytes/granulocytes because of the additional steps needed to purify them from the separation medium immediately following the blood separation. Our procedure involves blood collected in sodium citrate tubes, and it will be interesting to compare this method in terms of DNA quality and quantity from residual cells with blood collected in EDTA or heparin coated tubes. In summary, by demonstrating that substantial amounts of high quality DNA can be directly obtained from whole blood residue containing Ficoll solution, erythrocytes, and granulocytes, we have eliminated the need to purify the granulocytes prior to DNA extraction. This method offers laboratories and biobanks a flexible and cost-effective approach to obtain plasma, PBMCs, and large amounts of DNA from a single collection of blood for molecular genetics/epidemiologic studies.
We acknowledge Dr. Elizabeth R. Unger for her critical suggestions with the preparation of this manuscript. We also acknowledge Mr. Maung Khin for carrying out DNA extraction from a subset of the samples in this study.
The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the funding agency.
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