Name: rapid fractionation and isolation of whole blood components in samples obtained from a community-based setting
Uploaded: 2015-11-30T14:00:00
Description: Watch this Scientific Journal Video about Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting at JoVE.com

We would like to thank Henriette Mair-Meijers for invaluable attention to detail and hours devoted to processing the blood collections. We are grateful for the graphic design expertise of Natalie Jameson Kiesling. We also appreciate the approval of the manufacturers (Qiagen (Valencia, CA), BD Biosciences (San Jose, CA), Life Technologies (Grand Island, NY)) mentioned herein to publish the use of their products as described. Funding for this work was generously provided by the National Institutes of Health award numbers DA022720, RC1MH088283, and DA022720-05-S1.

The Detroit Neighborhood Health Study was reviewed and approved by the University of Michigan&#39;s Institutional Review Board. All participants provided informed consent prior to their participation in the study.
1. Overview
<ol>
	<li>Properly document all stages from recruitment through analysis of endpoint data. Perform the protocols on Day 1 simultaneously, switching stages and overlapping as time permits. The sequence of stages is written to optimize performance efficiency. Figu...

<ol>
	<li>Hernandez, M. E., Martinez-Fong, D., Perez-Tapia, M., Estrada-Garcia, I., Estrada-Parra, S., Pavon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effect+of+selective+serotonin-reuptake+inhibitors+on+lymphocyte+subsets+in+patients+with+a+major+depressive+disorder.">Evaluation of the effect of selective serotonin-reuptake inhibitors on lymphocyte subsets in patients with a major depressive disorder.</a> Eur Neuropsychopharmacol. 20, 88-95 (2010).</li><li>Weigelt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-1+and+DAP12+expression+in+monocytes+of+patients+with+severe+psychiatric+disorders+EGR3,+ATF3+and+PU.1+as+important+transcription+factors.">TREM-1 and DAP12 expression in monocytes of patients with severe psychiatric disorders EGR3, ATF3 and PU.1 as important transcription factors.</a> Brain Behav Immun. 25, 1162-1169 (2011).</li><li>Robertson, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+subset+differences+in+patients+with+chronic+fatigue+syndrome,+multiple+sclerosis+and+major+depression.">Lymphocyte subset differences in patients with chronic fatigue syndrome, multiple sclerosis and major depression.</a> Clin Exp Immunol. 141, 326-332 (2005).</li><li>Rotter, A., Asemann, R., Decker, A., Kornhuber, J., Biermann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orexin+expression+and+promoter-methylation+in+peripheral+blood+of+patients+suffering+from+major+depressive+disorder.">Orexin expression and promoter-methylation in peripheral blood of patients suffering from major depressive disorder.</a> J Affect Disord. 131, 186-192 (2011).</li><li>Klengel, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allele-specific+FKBP5+DNA+demethylation+mediates+gene-childhood+trauma+interactions.">Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions.</a> Nat Neurosci. 16, 33-41 (2013).</li><li>Segman, R. H., Shefi, N., Goltser-Dubner, T., Friedman, N., Kaminski, N., Shalev, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+blood+mononuclear+cell+gene+expression+profiles+identify+emergent+post-traumatic+stress+disorder+among+trauma+survivors.">Peripheral blood mononuclear cell gene expression profiles identify emergent post-traumatic stress disorder among trauma survivors.</a> Mol Psychiatry. 10, 500-513 (2005).</li><li>Smith, B. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cohort+Profile:+Generation+Scotland:+Scottish+Family+Health+Study+(GS:SFHS).+The+study,+its+participants+and+their+potential+for+genetic+research+on+health+and+illness.">Cohort Profile: Generation Scotland: Scottish Family Health Study (GS:SFHS). The study, its participants and their potential for genetic research on health and illness.</a> Int J Epidemiol. 42, 689-700 (2013).</li><li>Mallone, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+preservation+of+peripheral+blood+mononuclear+cells+for+analysis+of+islet+antigen-reactive+T+cell+responses:+position+statement+of+the+T-Cell+Workshop+Committee+of+the+Immunology+of+Diabetes+Society.">Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society.</a> Clin Exp Immunol. 163, 33-49 (2011).</li><li>Duvigneau, J. C., Hartl, R. T., Teinfalt, M., Gemeiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delay+in+processing+porcine+whole+blood+affects+cytokine+expression.">Delay in processing porcine whole blood affects cytokine expression.</a> J Immunol Methods. 272, 11-21 (2003).</li><li>Debey, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+different+isolation+techniques+prior+gene+expression+profiling+of+blood+derived+cells:+impact+on+physiological+responses,+on+overall+expression+and+the+role+of+different+cell+types.">Comparison of different isolation techniques prior gene expression profiling of blood derived cells: impact on physiological responses, on overall expression and the role of different cell types.</a> Pharmacogenomics J. 4, 193-207 (2004).</li><li>Uddin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+and+immune+function+profiles+associated+with+posttraumatic+stress+disorder.">Epigenetic and immune function profiles associated with posttraumatic stress disorder.</a> Proc Natl Acad Sci U S A. 107, 9470-9475 (2010).</li><li>Kessler, R. C., Wang, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+descriptive+epidemiology+of+commonly+occurring+mental+disorders+in+the+United+States.">The descriptive epidemiology of commonly occurring mental disorders in the United States.</a> Annu Rev Public Health. 29, 115-129 (2008).</li><li>. QIAamp&#174; DNA Mini and Blood Mini Handbook Available from: <a target="_blank" href="https://www.qiagen.com/us/resources/resourcedetail?id=67893a91-946f-49b5-8033-394fa5d752ea">https://www.qiagen.com/us/resources/resourcedetail?id=67893a91-946f-49b5-8033-394fa5d752ea</a> (2010)</li><li>Koenen, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SLC6A4+methylation+modifies+the+effect+of+the+number+of+traumatic+events+on+risk+for+posttraumatic+stress+disorder.">SLC6A4 methylation modifies the effect of the number of traumatic events on risk for posttraumatic stress disorder.</a> Depress Anxiety. 28, 639-647 (2011).</li><li>Walsh, K., Uddin, M., Soliven, R., Wildman, D. E., Bradley, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Associations+between+the+SS+variant+of+5-HTTLPR+and+PTSD+among+adults+with+histories+of+childhood+emotional+abuse:+Results+from+two+African+American+independent+samples.">Associations between the SS variant of 5-HTTLPR and PTSD among adults with histories of childhood emotional abuse: Results from two African American independent samples.</a> J Affect Disord. 161, 91-96 (2014).</li><li>Bustamante, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Childhood+maltreatment+is+associated+with+epigenetic+differences+in+hypothalamic-pituitary-adrenal+(HPA)+axis+genes+in+the+Detroit+Neighborhood+Health+Study.">Childhood maltreatment is associated with epigenetic differences in hypothalamic-pituitary-adrenal (HPA) axis genes in the Detroit Neighborhood Health Study.</a> , (2013).</li><li>Sipahi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+epigenetic+variation+of+DNA+methyltransferase+genes+is+associated+with+vulnerability+to+post-traumatic+stress+disorder.">Longitudinal epigenetic variation of DNA methyltransferase genes is associated with vulnerability to post-traumatic stress disorder.</a> Psychol Med. 44, 3165-3179 (2014).</li><li>Uddin, M., Koenen, K. C., Aiello, A. E., Wildman, D., de los Santos, R., Galea, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+and+inflammatory+marker+profiles+associated+with+depression+in+a+community-based+epidemiologic+sample.">Epigenetic and inflammatory marker profiles associated with depression in a community-based epidemiologic sample.</a> Psychol Med. 41, 997-1007 (2011).</li><li>Uddin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-traumatic+stress+disorder+is+associated+with+immunosenescent+T+cell+phenotypes+in+the+Detroit+Neighborhood+Health+Study.">Post-traumatic stress disorder is associated with immunosenescent T cell phenotypes in the Detroit Neighborhood Health Study.</a> , (2011).</li><li>Uddin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-traumatic+stress+disorder+is+associated+with+immunosenescent+T+cell+phenotypes+in+the+Detroit+Neighborhood+Health+Study.">Post-traumatic stress disorder is associated with immunosenescent T cell phenotypes in the Detroit Neighborhood Health Study.</a> , (2011).</li><li>Uddin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+signatures+of+post-traumatic+stress+disorder+in+the+Detroit+Neighborhood+Health+Study.">Biological signatures of post-traumatic stress disorder in the Detroit Neighborhood Health Study.</a> , (2010).</li><li>Aiello, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytomegalovirus+antibodies+as+a+marker+of+immunosenescence+in+the+Detroit+Neighborhood+Health+Study.">Cytomegalovirus antibodies as a marker of immunosenescence in the Detroit Neighborhood Health Study.</a> , (2010).</li><li>Bustamante, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Distinct gene expression profiles characterize lifetime PTSD and childhood maltreatment in the Detroit Neighborhood Health Study. , (2013).</li></ol>

We have described a streamlined protocol that has been successfully applied to process more than 1,600 whole blood samples in the Detroit Neighborhood Health Study. Although many of these techniques are available in the existing literature, our step-by-step compilation, including precisely timed alterations between each step, reflects an optimized, efficient protocol that successfully produces a variety of biologic specimens with a wide range of downstream applications, including DNA methylation, mRNA expression and immu...

Multiple studies have characterized differences in gene expression, DNA methylation and cell subset in blood among individuals with and without mental (or other) illnesses1-4. These studies, however, have been obtained from clinical settings in which disease-associated differences may be magnified due to the generally more severe nature of the illnesses for which patients are seeking treatment. Due to advances in &#8220;omics&#8221; approaches, the past decade has seen an explosion of interest in obtaining biologic samples from community and/or epidemiologic settings5-7, in order to provide population-based estimates of disease prevalence and a broader picture of the environmental determinants of these mental and/or physical illnesses.
A key challenge in this regard is the requirement for rapid processing of the collected specimens. Degradation of mononuclear cells, key immune system components that are frequently used to assess the health of an individual, begins immediately upon blood draw with a significant decrease in recovery after 2 hr of collection8-10. To address this challenge, we present an optimized protocol in which multiple components of human whole blood are simultaneously isolated from samples obtained in homes of subjects living in a large metropolitan area. The protocol is based upon our compilation and modification of current techniques, including storage of all &#8220;extra&#8221; fractions in the event future techniques allow for further isolation/analyses. While alternative methods or kits may be employed in place of the individual methods described here, those outlined have proven to be a reliable and efficient means for processing samples in a high-throughput manner. High-quality fractions (PBMCs, DNA, serum, and RNA) of fresh blood can be produced within 2 hr of collection and all assay-ready specimens can be available within 2 days (Figure 1).
This protocol was developed to enable the efficient processing of samples collected from community-dwelling, adult residents of the city of Detroit for testing in the Detroit Neighborhood Health Study (DNHS; DA022720, RC1MH088283, DA022720-05-S1), a population-based study of the social and biological determinants of post-traumatic stress disorder (PTSD) and other mental illnesses. The prevalence of PTSD in Detroit is more than twice the national average11,12. Identifying biological determinants of PTSD in this population may help to develop appropriate pharmacologic and/or cognitive-behavioral interventions to aid those suffering from the disorder, both in this urban population, and in other high-risk populations (e.g., returning military veterans). Our laboratory, previously located at Wayne State University in Detroit, Michigan, was selected for processing based on our expertise in handling fresh tissue samples derived from a variety of sources, the necessity to begin processing the samples within 2 hr of collection, and our proximity to the collection sites. With this unique opportunity at hand, our goal was to optimize the processing for greatest yield of DNA, RNA, serum and peripheral blood mononuclear cells (PBMCs) from each specimen (a total of N=1,639 samples over 5 waves of specimen collection). The procedures outlined here can be performed simultaneously in a non-clinical setting, thus producing starting material (see Table 1 for average yields) for a multitude of downstream applications including microarray, epigenetic, real-time RT-PCR, and flow cytometry analyses.
<img alt="Figure 1" src="/files/ftp_upload/52227/52227fig1highres.jpg" width="700" /> 
Figure 1. Overall work flow. The overall process depicted here includes the logistics of obtaining the blood specimens from identifying consenting participants to the blood draw itself. High-quality, fractions (peripheral blood mononuclear cells; PBMCs, DNA, serum, and RNA) of fresh whole blood can be produced within 2 hr of collection and all assay-ready specimens can be available within 2 days. Moreover, the fractions prepared through this method are suitable for long-term storage if samples are not to be tested immediately. The entire timeline outlined here could be completed in a single day (~5 hr total). However, such a day would be extremely labor intensive especially for a single technician with substantial experience with the techniques. Thus, we recommend dividing the procedures on Day 1 between at least two technicians and completing the RNA processing on Day 2. <a href="https://www.jove.com/files/ftp_upload/52227/52227fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>QIAamp DNA Blood Mini Kit</td>
			<td>Qiagen</td>
			<td>51104</td>
			<td>Day 1: DNA isolation</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS)</td>
			<td>Sigma</td>
			<td>P5493-1L</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>5 &#190;&#8221; Pasteur pipets</td>
			<td>Fisher</td>
			<td>13-678-6A</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), heat inactivated</td>
			<td>Life Technologies</td>
			<td>10082147</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma</td>
			<td>D8418-500ml</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>RPMI Medium 1640, liquid</td>
			<td>Invitrogen</td>
			<td>11875119</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>0.4% trypan blue stain&#160;</td>
			<td>Invitrogen</td>
			<td>T10282</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter or cell counting device such as a microscope and hemocytometer</td>
			<td>Invitrogen</td>
			<td>C10281</td>
			<td>Day 1: PBMC isolation</td>
		</tr>
		<tr>
			<td>LeukoLOCK Fractionation &#38; Stabilization Kit&#160;</td>
			<td>Ambion</td>
			<td>1933</td>
			<td>Day 1: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>25G x 5/8 in. needles</td>
			<td>Becton Dickinson</td>
			<td>305122</td>
			<td>Day 1: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>Syringes (5ml)</td>
			<td>Becton Dickinson</td>
			<td>309646</td>
			<td>Days 1 and 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>Denaturing Lysis Solution&#160;</td>
			<td>Ambion</td>
			<td>8540G</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>5M NaCl</td>
			<td>Life Technologies</td>
			<td>24740011</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>TRI Reagent&#160;</td>
			<td>Ambion</td>
			<td>9738</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>Bromo-3-chloro-propane (BCP)</td>
			<td>Sigma</td>
			<td>B-9673</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>spin cartridges&#160;</td>
			<td>Ambion</td>
			<td>10051G</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>0.1mM EDTA</td>
			<td>Ambion</td>
			<td>9912</td>
			<td>Day 2: Leukocyte RNA isolation</td>
		</tr>
		<tr>
			<td>DNA-free Kit</td>
			<td>Ambion</td>
			<td>AM1960</td>
			<td>Day 2: DNase treament</td>
		</tr>
		<tr>
			<td>RNA 6000 Ladder&#160;</td>
			<td>Agilent</td>
			<td>5067-1529</td>
			<td>Day 2: Bioanalyzer analysis</td>
		</tr>
		<tr>
			<td>RNA 6000 Nano Series II Kit&#160;</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td>Day 2: Bioanalyzer analysis</td>
		</tr>
		<tr>
			<td>RNaseZAP</td>
			<td>Ambion</td>
			<td>AM8782</td>
			<td>Day 2: Bioanalyzer analysis</td>
		</tr>
		<tr>
			<td>Ethanol &#62;99%</td>
			<td>Sigma</td>
			<td>E7023-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol &#62;99%&#160;</td>
			<td>Sigma</td>
			<td>I9516-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free ultra pure water&#160;</td>
			<td>Invitrogen</td>
			<td>9938</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips (nuclease-free)</td>
			<td>Eppendorf</td>
			<td>22491253</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetter (serological)</td>
			<td>Eppendorf</td>
			<td>2223020-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetters (for volumes under 1ml)</td>
			<td>Eppendorf</td>
			<td>3120000-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes (serological)</td>
			<td>Fisher</td>
			<td>13-678-27E</td>
			<td></td>
		</tr>
		<tr>
			<td>Controlled rate freezing containers</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryoboxes (to hold 2ml and 5ml cryovials and 1.5ml microcentrifuge tubes)</td>
			<td>Fisher</td>
			<td>03-395-464</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tube rack</td>
			<td>Thermo Scientific</td>
			<td>14-804-134</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml polypropylene tubes&#160;</td>
			<td>Fisher</td>
			<td>14-959-49D</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml and 0.65 microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>07-200-534 and 07-200-185</td>
			<td></td>
		</tr>
		<tr>
			<td>2ml and 5ml cryovials</td>
			<td>Fisher</td>
			<td>10-500-26 and 10-269-88F&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>8ml CPT vacutainer</td>
			<td>BD Biosciences</td>
			<td>362761</td>
			<td>2 tubes</td>
		</tr>
		<tr>
			<td>6ml K2 EDTA vacutainer</td>
			<td>BD Biosciences</td>
			<td>367863</td>
			<td>2 tubes</td>
		</tr>
		<tr>
			<td>8.5ml SST vacutainer</td>
			<td>BD Biosciences</td>
			<td>367988</td>
			<td>1 tube</td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Fisher</td>
			<td>2215365</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry bath incubator with heating block for microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>11-715-1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Filtration/vacuum system for use within the cell culture hood</td>
			<td>Fisher</td>
			<td>01-257-87</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed-angle rotor for microcentrifuge tubes with aerosol-tight lid</td>
			<td>Eppendorf</td>
			<td>22637002</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge with a swing-bucket rotor and aerosol-tight caps for 16 x 125mm vacutainers and 15ml polypropylene tubes&#160;</td>
			<td>Eppendorf</td>
			<td>22628157</td>
			<td>2, one does not need to be refrigerated</td>
		</tr>
		<tr>
			<td>Nanodrop 2000 (recommended for accuracy of small volumes) or other spectrophotometric device</td>
			<td>Fisher</td>
			<td>13-400-411</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen tank</td>
			<td>Thermo Scientific</td>
			<td>11-676-56</td>
			<td></td>
		</tr>
		<tr>
			<td>-80 &#730;C freezer</td>
			<td>Thermo Scientific</td>
			<td>992RAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharps container</td>
			<td>Fisher</td>
			<td>22-037-970</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological waste container</td>
			<td>Thermo Scientific</td>
			<td>1223P52</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Level 2&#160; certified cell culture hood</td>
			<td>Thermo Scientific</td>
			<td>13-261-315</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid fractionation and isolation of whole blood components in samples obtained from a community-based setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

It is essential that the overall procedure produce high quality material for analysis in a multitude of downstream applications including gene expression via microarray and RT-PCR analysis, detection of epigenetic modifications, and cell subset variations. Table 1 indicates the average yield and quality of materials from each of the processes. Figure 3 provides an example of the quality output of the leukocyte RNA isolation and filter processing methods. The image on the upper left of <s...

Watch this Scientific Journal Video about Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting at JoVE.com

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

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