This work was supported, in whole or in part, by NIH Grants R01 AA 020758-04, U01DK 061731-13 and T32 DK 007150-38 to AJS and T32 DK 007150-38 to AA. This is original work and is not under consideration elsewhere for publication.

1. Collection of Blood Samples
<ol>
	<li>Collect fasting peripheral venous blood samples into 10 ml plastic tubes containing anticoagulant Ethylenediaminetetraacetic acid (EDTA) (which has several advantages over other anticoagulants) by standard venipuncture of a prominent vein in the antecubital fossa.</li>
	<li>Centrifuge the blood samples at 1,600 x g for 20 min at 4 &#176;C in a tabletop centrifuge to obtain plasma free of red blood cells and small amounts of RNA.</li>
	<li>Sequentially centrifuge the supernatant ...

<ol>
	<li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116 (2), 281-297 (2004).</li><li>Arroyo, J. D., 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=Argonaute2+complexes+carry+a+population+of+circulating+microRNAs+independent+of+vesicles+in+human+plasma.">Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma.</a> Proc Natl Acad Sci U S A. 108 (12), 5003-5008 (2011).</li><li>Vickers, 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=MicroRNAs+are+transported+in+plasma+and+delivered+to+recipient+cells+by+high-density+lipoproteins.">MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins.</a> Nat Cell Biol. 13 (4), 423-433 (2011).</li><li>Wagner, 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=Characterization+of+levels+and+cellular+transfer+of+circulating+lipoprotein-bound+microRNAs.">Characterization of levels and cellular transfer of circulating lipoprotein-bound microRNAs.</a> Arterioscler Thromb Vasc Biol. 33, 1392-1400 (2013).</li><li>Wang, 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=MicroRNAs+185,+96,+and+223+repress+selective+high-density+lipoprotein+cholesterol+uptake+through+posttranscriptional+inhibition.">MicroRNAs 185, 96, and 223 repress selective high-density lipoprotein cholesterol uptake through posttranscriptional inhibition.</a> Mol Cell Biol. 33 (10), 1956-1964 (2013).</li><li>Rayner, K. J., Moore, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+control+of+high-density+lipoprotein+metabolism+and+function.">MicroRNA control of high-density lipoprotein metabolism and function.</a> Circ Res. 114 (1), 183-192 (2014).</li><li>Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes:+endosomal-derived+vesicles+shipping+extracellular+messages.">Exosomes: endosomal-derived vesicles shipping extracellular messages.</a> Curr Opin Cell Biol. 16 (4), 415-421 (2004).</li><li>Redgrave, T. G., Roberts, D. C., West, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+plasma+lipoproteins+by+density-gradient+ultracentrifugation.">Separation of plasma lipoproteins by density-gradient ultracentrifugation.</a> Anal Biochem. 65, 42-49 (1975).</li><li>Foreman, J. 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=Fractionation+of+human+serum+lipoproteins+by+single-spin+gradient+ultracentrifugation:+quantification+of+apolipoproteins+B+and+A-1+and+lipid+components.">Fractionation of human serum lipoproteins by single-spin gradient ultracentrifugation: quantification of apolipoproteins B and A-1 and lipid components.</a> J Lipid Res. 18, 759-767 (1977).</li><li>Dong, 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=Serum+LDL-+and+HDL-cholesterol+determined+by+ultracentrifugation+and+HPLC.">Serum LDL- and HDL-cholesterol determined by ultracentrifugation and HPLC.</a> J Lipid Res. 52, 383-388 (2011).</li><li>Tong, H., Knapp, H. R., VanRollings, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=low+temperature+flotation+method+to+rapidly+isolate+lipoproteins+from+plasma.">low temperature flotation method to rapidly isolate lipoproteins from plasma.</a> J Lipid Res. 39, 1696-1704 (1998).</li><li>Fless, G. M., ZumMallen, M. E., Scanu, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physicochemical+properties+of+apolipoprotein+(a)+and+lipoprotein+(a-)+derived+from+the+dissociation+of+human+plasma+lipoprotein+(a).">Physicochemical properties of apolipoprotein (a) and lipoprotein (a-) derived from the dissociation of human plasma lipoprotein (a).</a> J Biol Chem. 261, 8712-8718 (1986).</li><li>Brownie, 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=The+elimination+of+primer-dimer+accumulation+in+PCR.">The elimination of primer-dimer accumulation in PCR.</a> Nucleic Acids Res. 25, 3235-3241 (1997).</li><li>Alton, E., Inyoul, L., Leroy, H., David, G., Kai, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+microRNA:+a+new+source+of+biomarkers.">Extracellular microRNA: a new source of biomarkers.</a> Mutat Res. 717 (1-2), 85-90 (2011).</li><li>Stefanie, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+biomarker+profiling+with+microRNAs.">Cancer biomarker profiling with microRNAs.</a> Nature Biotechnology. 26, 400-401 (2008).</li><li>Prasun, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+as+promising+biomarkers+in+cancer+diagnostics.">MicroRNAs as promising biomarkers in cancer diagnostics.</a> Biomarker Research. 2 (19), (2014).</li><li>Creemers, E. E., Tijsen, A. J., Pinto, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs:+novel+biomarkers+and+extracellular+communicators+in+cardiovascular+disease?.">Circulating microRNAs: novel biomarkers and extracellular communicators in cardiovascular disease?.</a> Circ Res. 110, 483-495 (2012).</li><li>Jonathan, S., Martin, S., Eric, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> miRNA profiling from blood -challenges and recommendations. , (2015).</li><li>Francesco, M., Paola, D. C., Anna, T., Jesper, T., Sergio, A., Riccardo, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+of+circulating+microRNA+expression+data+obtained+by+quantitative+real-time+RT-PCR.">Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR.</a> Brief Bioinform. 3, 1-9 (2015).</li><li>Chen, Y., 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=Circulating+microRNAs,+novel+biomarkers+of+acute+myocardial+infarction:+a+systemic+review.">Circulating microRNAs, novel biomarkers of acute myocardial infarction: a systemic review.</a> World J Emerg Med. 3, 257-260 (2012).</li></ol>

The authors have nothing to disclose.

Identification of novel biomarkers from blood will aid in the clinical diagnosis and prognosis of various diseases. MicroRNAs have known to possess all the qualities of biomarkers and have been shown in various studies 14-17. In this study we have demonstrated rapid and simple easy method to isolate miRNA from plasma HDL. Conventional density gradient ultra-centrifugation method of isolation of VLDL, LDL and HDL depends on accurate sampling of plasma, precise preparation of the buffer solution, measurement of ...

MicroRNAs are endogenous non-coding tiny RNA species that are highly conserved and are considered key players in the regulation of various biological processes by degrading or repressing specific target messenger RNAs1. Because miRNAs act intracellularly they have been explored as tissue-derived biomarkers which led to the discovery of tissue-specific functions of these miRNA. However, miRNAs are also found extracellularly either associated with proteins or in exosomes/micro vesicles that effectively can shield them from degradation by extracellular RNases2. More recent studies have shown that the protective effect of HDL may not be closely linked to its capability to promote cholesterol efflux but rather to its non-cholesterol cargo, in particularly as a circulating miRNAs carrier 3, 4. These miRNAs may not only modulate lipid metabolism but are also associated with anti-inflammatory, antioxidant and antithrombotic effects of the HDL-miRNA complex 5, 6.
To further explore the role of miRNAs carried in HDL particles, a simple and easy protocol needs to be established for miRNA extraction from isolated highly purified HDL for use in clinical routine. Numerous methods have been described to isolate HDL. These methods are either very time consuming or require large volume of plasma that may require sample pooling, extensive dialysis for desalting isolated lipoproteins and they do not completely remove exosomes as a source of miRNAs3, respectively. Here we describe a simple and rapid method that can isolate miRNA from highly purified HDL utilizing small volume of blood samples on a larger scale. We believe that this method may serve as good reference to promote research into the role of circulating miRNAs and in particular the role of HDL in facilitating communication between various cells and organs.

<table border="0" cellpadding="0" cellspacing="0" width="965">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">Plastic Vacutainer Lavender K2EDTA tubes&#160;</td>
			<td style="width:279px;">Becton, Dickinson and Company</td>
			<td style="width:172px;">366643</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Thermo Scientific, Sorvall Legend X1R&#160;</td>
			<td style="width:172px;">75004261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Densito 30PX densitometer</td>
			<td>Mettler Toledo</td>
			<td style="width:172px;">MT51324450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ExoQuick solution&#160;</td>
			<td>Invitrogen</td>
			<td style="width:172px;">4484451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polycarbonate thick-walled ultracentrifuge tube</td>
			<td>Thermo Scientific</td>
			<td style="width:172px;">O3237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorvall WX100 ultracentrifuge&#160;</td>
			<td>Thermo Scientific</td>
			<td style="width:172px;">46902</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fat Red 7B&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:172px;">201618</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-mercaptoethanol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15 Centrifugal filter devices 10K</td>
			<td>Millipore</td>
			<td style="width:172px;">UFC901008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-centrifugal filter devices 3K</td>
			<td>Millipore</td>
			<td style="width:172px;">UFC800308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickGel Lipo kit&#160;</td>
			<td>Helena Laboratories&#160;</td>
			<td style="width:172px;">3344,3544T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human lipoprotein standards for VLDL, LDL and HDL</td>
			<td>LipoTrol; Helena Laboratories</td>
			<td style="width:172px;">5069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rep Prep buffer&#160;</td>
			<td>Helena Laboratories&#160;</td>
			<td style="width:172px;">3100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy MinElute spin columns&#160;</td>
			<td>Qiagen</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop 1000 analyzer</td>
			<td>Thermo Scientific</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">miScript II RT Kit&#160;</td>
			<td>Qiagen</td>
			<td style="width:172px;">218161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CFX96 Touch real-time PCR detection system</td>
			<td>BioRad</td>
			<td style="width:172px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miRNeasy Serum/Plasma Kit</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">217184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miScript Primer Assays</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">141078139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miScript SYBR Green PCR Kit&#160;</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">218073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:348px;">miRNeasy Serum/Plasma Spike-In Control</td>
			<td style="width:279px;">QIAGEN</td>
			<td style="width:172px;">219610</td>
			<td></td>
		</tr>
	</tbody>
</table>

fast and simplified method for high through-put isolation of mirna from highly purified high density lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

Isolation of High Density Lipoprotein After Removal of Exosomes 
To obtain miRNA from highly purified HDL it is necessary to remove exosomes that represent a source of miRNA contamination7. This was done prior to density gradient ultracentrifugation with a commercially available kit. For practical purposes a three step standard density gradient ultracentrifugation protocol developed by commercial company was modified (Figure 1</str...

Watch this Scientific Journal Video about Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein at JoVE.com

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...

The overall goal of this method is to develop a simplified approach to rapidly isolate purified high density lipoproteins from human blood plasma for micro RNA analysis. This method can help answer key questions in hepatology and cardiology, such as understanding the molecular mechanisms and protective functions of high density lipoproteins. Some main advantages of this technique is that purified HDL micro RNA come pre-isolated from less volume of samples within a short period of time.In this protocol, plasma is obtained from fasting peripheral venous blood samples by multiple centrifugation steps as described in the protocol text. Transfer the plasma to a new tube. And measure the density of the plasma using a densitometer at room temperature, per the manufacturer's instructions.To remove the circulating exosomes that represent a source of micro RNA contamination, add 252 microliters of exosome precipitation solution to one milliliter of plasma. And incubate for 30 minutes at four degrees Celsius. Then centrifuge the mixture for 30 minutes to pellet out the exosomes.Transfer one milliliter of the resulting supernatant to a polycarbonate thick walled ultracentrifuge tube for further processing, as demonstrated in the next video segment. Isolation of high density lipoproteins, or HDL, is accomplished by a three step density gradient ultracentrifugation protocol, that involves a sequential isolation of very low density lipoproteins, or VLDL, low density lipoproteins, or LDL, and HDL. The three different density solutions, A, B, and C, must be prepared fresh, following the procedure described in the protocol text.In a 6.5 milliliter polycarbonate thick walled ultracentrifuge tube, mix one milliliter of plasma and 200 microliters of nuclease-free Fat Red 7B. Then, carefully layer five milliliters of solution A on top of the mixture. If needed, add additional Fat Red 7B on top of solution A to balance the weight of each tube.Load the tubes into a pre-chilled rotor. Place the rotor into a floor centrifuge and centrifuge for two hours. At the end of the run, two layers should be visible.Remove 1.5 milliliters of the top layer, representing the VLDL fraction. And store it four degrees Celsius. Use a pipette to transfer four milliliters from the bottom of the tube to a new 50 milliliter tube.This contains the LDL and HDL fraction. The next step is the isolation of LDL. Mix two milliliters of solution B and 100 microliters of nuclease-free Fat Red 7B into the tube containing the LDL and HDL fraction.Put on ice for five to ten minutes. Transfer 6.5 milliliters of the mixed sample into a polycarbonate thick walled ultracentrifuge tube. Load the tube into a pre-chilled rotor and centrifuge for three hours.When the centrifugation is complete, remove 1.5 milliliters of the top layer, representing the LDL fraction and keep at four degrees Celsius. Transfer four milliliters from the bottom of the tube, containing the HDL fraction to a 50 milliliter sterile tube. The third step is the isolation of HDL.Mix two milliliters of solution C, 100 microliters of nuclease-free Fat Red 7B, and 15 microliters of 98%beta mercaptoethanol into the tube, containing the HDL fraction. Check the density of the mixture. Load the tube into a pre-chilled rotor and centrifuge for three hours.After that, remove two milliliters of the top layer, representing the HDL fraction, and keep at four degrees Celsius. Excessive salt must be removed from the lipoprotein fractions to avoid interference with subsequent agarose gel electrophoresis and PCR. In this video, desalting and concentration will be demonstrated for only the HDL fraction.Add 13 milliliters of cold PBS to the HDL fraction and transfer to a centrifugal filter device with the molecular weight cut-off of 10K. Centrifuge in a swinging bucket rotor, at 4000 times g, and four degrees Celsius, for 30 minutes. Desalt the HDL fraction, a second time, using 13 milliliters of cold PBS, and centrifuging as before.After centrifugation, remove the lipoprotein containing solutes, and keep at four degrees Celsius. To assess the quality and purity of the isolated lipoproteins, agarose gel electrophoresis was performed with human lipoprotein standards, included as a size reference. Representative results from an isolation, without beta mercaptoethanol, showed that the HDL fraction is devoid of any contamination of VLDL and exhibits typical alpha mobility.However, the HDL fraction also shows traces of beta mobility due to contamination with lipoproteins. The addition of beta mercaptoethanol to solution C, during the last centrifugation step, effectively removes all contaminating lipoproteins from the HDL fraction. To demonstrate the feasibility of quantifying micro RNAs, carried in human HDL, purified with this method, two different micro RNAs were amplified by real-time quantitative PCR.In these amplification plots, the horizontal lines represent the detection threshold. Representative real-time PCR data from isolated HDL, obtained from six samples, showed the consistent detection of both micro RNAs. Lastly, melting curve analyses clearly show a distinct single peak for both micro RNAs, consistent with specific amplification in the preceding PCR.Once mastered, this technique can be done in nine hours, together with the desalting procedures, if it is done properly. While attempting this procedure, it is important to work at four to eight degrees centigrade, at all times. And to adjust the density of each lipoprotein fraction before ultracentrifugation.Following this procedure, other methods, such as electron microscopy, can be performed in order to answer additional questions, like the purity of HDL. After its development, this technique will allow researchers, in the field of metabolic syndrome, to explore HDL-associated micro RNAs as bio markers, in humans with suspected fatty liver disease. It may also be used to better understand the function of HDL and the molecular mechanisms by which it alters cell biology.

fast-simplified-method-for-high-through-put-isolation-mirna-from

Research

JoVE Journal

Biology

11.8K visualizações.  Virginia Commonwealth University. The overall goal of this method is to develop a simplified approach to rapidly isolate purified high density lipoproteins from human blood plasma for micro RNA analysis. This method can help answer key questions in hepatology and cardiology, such as understanding the molecular mechanisms and protective functions of high density lipoproteins. Some main advantages of this technique is that purified HDL micro RNA come pre-isolated from less volume of samples within a short period of time....