Name: identification of coding and non-coding rna classes expressed in swine whole blood
Uploaded: 2018-11-28T14:30:00
Description: Watch this Scientific Journal Video about Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood at JoVE.com

This work was mainly supported by the by USDA NIFA AFRI 2013-67015-21236, and in part by USDA NIFA AFRI 2015-67015-23216. This study was supported in part by an appointment to the Agricultural Research Service Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the US Department of Energy (DOE) and the US Department of Agriculture. ORISE is managed by Oak Ridge Associated Universities under DOE contract no. DE-AC05-06OR2310.
We would like to thank Dr. Kay Faaberg for the HP-PRRSV infectious clones, Dr. Susan Brockmeier for her help with animals involved in the experiment, and Sue Ohlendorf for secretarial assistance in preparation of the manuscript.

Animal protocols were approved by the National Animal Disease Center (USDA-ARS-NADC) Animal Care and Use Committee.
1. Collection of Swine Blood Samples
<ol>
	<li>Collect blood samples into RNA tubes. Collect ~2.5 mL or more if larger collection tubes are available.</li>
</ol>
2. Processing of Swine Blood Samples
<ol>
	<li>Centrifuge the blood tubes at 5,020 x g for 10 min at room temperature (15-25 &#176;C). If processing frozen samples incubate tube at ro...

<ol>
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N., Heike, G., Martin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+RNA+Depletion+for+Efficient+Use+of+RNA-Seq+Capacity.">Ribosomal RNA Depletion for Efficient Use of RNA-Seq Capacity.</a> Current Protocols in Molecular Biology. 103 (1), 11-14 (2013).</li><li>Fleming, D. S., Miller, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+small+non-coding+RNA+classes+expressed+in+swine+whole+blood+during+HP-PRRSV+infection.">Identification of small non-coding RNA classes expressed in swine whole blood during HP-PRRSV infection.</a> Virology. 517, 56-61 (2018).</li><li>Fleming, D. S., Miller, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+non-coding+RNA+expression+status+in+animals+faced+with+highly+pathogenic+porcine+reproductive+and+respiratory+syndrome+virus+(HP-PRRSV).">Small non-coding RNA expression status in animals faced with highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV).</a> Proceedings of the World Congress on Genetics Applied to Livestock Production. (11), (2018).</li><li>Bivens Nathan, J., Zhou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq+Library+Construction+Methods+for+Transcriptome+Analysis.">RNA-Seq Library Construction Methods for Transcriptome Analysis.</a> Current Protocols in Plant Biology. 1 (1), 197-215 (2016).</li><li>Cui, P., 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+comparison+between+ribo-minus+RNA-sequencing+and+polyA-selected+RNA-sequencing.">A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing.</a> Genomics. 96 (5), 259-265 (2010).</li><li>Guo, 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=RNAseq+by+Total+RNA+Library+Identifies+Additional+RNAs+Compared+to+Poly(A)+RNA+Library.">RNAseq by Total RNA Library Identifies Additional RNAs Compared to Poly(A) RNA Library.</a> BioMed Research International. 2015, 9 (2015).</li><li>Choi, I., 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=Increasing+gene+discovery+and+coverage+using+RNA-seq+of+globin+RNA+reduced+porcine+blood+samples.">Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples.</a> BMC Genomics. 15, 954 (2014).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TruSeq&#174;+Stranded+Total+RNA+Sample+Preparation+Guide.">TruSeq&#174; Stranded Total RNA Sample Preparation Guide.</a> Illumina. , (2018).</li><li>. New England Biolabs Inc. Protocol for use with NEBNext Multiplex Small RNA Library Prep Kit for Illumina (Index Primers 1-48) (E7560) Available from: <a target="_blank" href="https://www.neb.com/~/media/Catalog/All-Protocols/758F75A29CDE4D03954E1EF75E78EA3D/Content/manualE7560_Figure_1.jpg">https://www.neb.com/~/media/Catalog/All-Protocols/758F75A29CDE4D03954E1EF75E78EA3D/Content/manualE7560_Figure_1.jpg</a> (2018)</li><li>Shin, 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=Variation+in+RNA-Seq+transcriptome+profiles+of+peripheral+whole+blood+from+healthy+individuals+with+and+without+globin+depletion.">Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion.</a> PLoS One. 9 (3), 91041 (2014).</li><li>Costa, V., Angelini, C., De Feis, I., Ciccodicola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+the+complexity+of+transcriptomes+with+RNA-Seq.">Uncovering the complexity of transcriptomes with RNA-Seq.</a> Journal of Biomedicine and Biotechnology. 2010, 853916 (2010).</li><li>Martens-Uzunova, E. 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The first critical step in the protocol that made it optimized included the added globin depletion steps, which made it possible to get quality reads from whole blood samples. One of the largest limitations on using whole blood in sequencing studies are the high numbers of reads in the sample that will map to globin molecules and reduce the reads that could map to other molecules of interest18. Therefore, in optimizing the protocol for our sample type, we needed to incorporate a globin depletion s...

Next generation sequencing (NGS) has emerged as a powerful tool for the investigation of the changes that occur at the genomic level of biological organisms. Sample preparation for NGS methods can be varied depending on the organism, tissue type, and more importantly the questions the researchers are keen to address. Many studies have turned to NGS as a means of studying the differences in gene expression between states such as healthy and sick individuals1,2,3,4. The sequencing take place on a whole genome basis and allows a researcher to capture the most, if not all, of the genomic information for a particular genetic marker at a time point.
The most common markers of expression observed are the messenger RNAs (mRNAs). The most used procedures for prepping libraries for RNA-seq are optimized for the recovery of mRNA molecules through the use of a series of purifications, fragmentations, and ligations5,6. However, the decision on how a protocol is to be performed relies heavily on the sample type and the questions being posed about said sample. In most cases total RNA is extracted; yet, not all RNA molecules are of interest and in cases such as mRNA expression studies overly abundant RNA species, like ribosomal RNAs (rRNA) need to be removed to increase the number of detectable transcripts associated with the mRNAs. The most popular and widely used method for removing the abundant rRNA molecules is the reduction of polyadenylated RNA transcripts referred to as polyA depletion7. This approach works well for the analysis of mRNA expression as it does not affect the mRNA transcripts. However, in studies that are interested in non-coding or viral RNAs, polyA depletion also removes these molecules.
Many studies choose to focus on the RNA sequence library preparation to examine either mRNA expression (coding) or a particular class of small or large non-coding RNA. Although there are other procedures8 like ours that allow for the dual sample preparation, many studies prepare libraries from separate samples for separate studies when available. For a study like ours, this would normally require multiple blood samples increasing time, consumables, and animal stress. The goal of our study was to be able to use whole blood from animals to identify and quantify the different classes of both mRNA and non-coding RNA expressed between healthy and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) challenged pigs9,10 despite having only a single whole blood sample (2.5 mL) from each pig. In order to do this, we needed to optimize the typical extraction and library creation protocols to generate the proper data to allow for analysis of both mRNA and non-coding RNA (ncRNA) expression11 from a single sample.
This prompted a need for a protocol that allowed for mRNA and non-coding RNA analysis because the available standard kits and methods for RNA-extraction and library creation were intended chiefly for mRNA and use a poly-A depletion step12. This step would have made it impossible to recover non-coding RNA or viral transcripts from the sample. Therefore, an optimized method was needed that allowed for total RNA extraction without sample polyA depletion. The method presented in this manuscript has been optimized to allow for the use of whole blood as a sample type and to build sequencing libraries for both mRNA and ncRNAs of small and large sizes. The method has been optimized to allow for the analysis of all detectable non-coding RNAs as well as retain viral RNAs for later investigation13. In all, our optimized library preparation protocol allows for the investigation of multiple RNA molecules from a single whole blood sample.
The overall goal behind the use of this method was to develop a process that allowed for the collection of both non-coding RNA and mRNA from one sample of whole blood. This allows us to have mRNA, ncRNA, and viral RNA for each animal in our study sourced from a single sample9. This, ultimately, allows for more scientific discovery without additional animal costs and gives a more complete picture of the expression of each individual sample. The described method allows for the examination of the regulators of gene expression as well as allowing for completion of correlative studies comparing both mRNA and non-coding RNA expression using a single whole blood sample. Our study used this protocol to examine the changes in gene expression and possible epigenetic regulators in virally infected 9-week old male commercial pigs.

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			<td height="21" style="height:21px;width:319px;">0.2 mL thin-walled tubes</td>
			<td style="width:159px;">ThermoFisher</td>
			<td style="width:131px;">98010540</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:319px;">1.5 mL RNase/DNase - free tubes</td>
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			<td height="21" style="height:21px;width:319px;">Globin Reduction Oligo (&#945; 1)</td>
			<td style="width:159px;">Any supplier</td>
			<td style="width:131px;"></td>
			<td>Sequence GAT CTC CGA GGC TCC AGC TTA ACG GT</td>
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			<td height="21" style="height:21px;width:319px;">Globin Reduction Oligo (&#945; 2)</td>
			<td style="width:159px;">Any supplier</td>
			<td style="width:131px;"></td>
			<td>Sequence TCA ACG ATC AGG AGG TCA GGG TGC AA</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Globin Reduction Oligo (&#946; 1)</td>
			<td style="width:159px;">Any supplier</td>
			<td style="width:131px;"></td>
			<td>Sequence AGG GGA ACT TAG TGG TAC TTG TGG GT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Globin Reduction Oligo (&#946; 2)</td>
			<td style="width:159px;">Any supplier</td>
			<td style="width:131px;"></td>
			<td>Sequence GGT TCA GAG GAA AAA GGG CTC CTC CT</td>
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			<td height="21" style="height:21px;width:319px;">10X Oligo Hybridization Buffer</td>
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			<td height="21" style="height:21px;width:319px;">&#160;-Tris-HCl, pH 7.6</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:131px;">BP1757-100</td>
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			<td height="21" style="height:21px;width:319px;">Agilent RNA 6000 Nano Kit</td>
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			<td height="21" style="height:21px;width:319px;">TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero&#160;</td>
			<td style="width:159px;">Illumina</td>
			<td style="width:131px;">RS-122-2201</td>
			<td>mRNA kit; Human/Mouse/Rat Set A&#160; (48 samples, 12 indexes)</td>
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			<td height="21" style="height:21px;width:319px;">TruSeq Stranded Total RNA Sample Preparation Guide</td>
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			<td style="width:131px;"></td>
			<td>Available on-line</td>
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			<td height="21" style="height:21px;width:319px;">MicroAmp Optical 96 well plates</td>
			<td style="width:159px;">ThermoFisher</td>
			<td style="width:131px;">N8010560</td>
			<td>These were used in place of .3mL plates as needed</td>
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			<td height="42" style="height:42px;width:319px;">NEBNext Multiplex Small RNA Library Prep Set for Illumina&#174; (Set 1)&#160;</td>
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			<td style="width:131px;">E73005</td>
			<td>small RNA kit</td>
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			<td height="42" style="height:42px;width:319px;">NEBNext Multiplex Small RNA Library Prep Set for Illumina&#174; (Set 2)&#160;</td>
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			<td>small RNA kit</td>
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			<td height="21" style="height:21px;width:319px;">QIAQuick PCR Purification Kit</td>
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identification of coding and non-coding rna classes expressed in swine whole blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

The representative samples in our study are the globin and ribo-depleted whole blood samples. The representative outcome of the protocol consists of a globin depleted library sample with an RNA integrity number (RIN) above 7 (Figure 1a) and 260/280 nm concentration ratios at or above 2 (Figure 1b and 1c). Validation of the sample outcome was performed using spectrophotometer to give the final con...

Watch this Scientific Journal Video about Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood at JoVE.com

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

This method can help answer key questions in the study of host-pathogen interactions by allowing examination of the coding, non-coding, and viral RNA expression involved in the host amino response as well as how a pathogen can affect the host's biological functions. The main advantage of this technique is it presents a protocol optimized for the processing of mRNA and non-coding RNA from globin-reduced RNAseq libraries from a single whole blood sample. Demonstrating the technique will be Sarah Anderson, a technician from my laboratory.Start by centrifuging the blood tubes at 50 20 times G for 10 minutes at room temperature. Working in a biological safety cabinet, after removing the supernatant, add eight milliliters of RNase-free water to the pellet. Close the tubes and vortex the pellet until it is visibly dissolved, then centrifuge the tubes at 50 20 times G for 10 minutes at room temperature to recover the pellet.Working in a biological safety cabinet, discard the supernatant and save the pellet. To isolate total RNA, start by pipetting 300 microliters of lysis binding buffer to the pellet. After vortexing, transfer the mixture from each tube to a new labeled 1.5 milliliter centrifuge tube.Add 30 microliters of homogenate additive from the miRNA isolation kit. Vortex the tubes and place on the ice for 10 minutes. Working in a fume hood, remove the tubes from ice and add 300 microliters of the acid phenol chloroform reagent from the kit and vortex again to mix.After centrifuging at 10, 000 times G for five minutes at room temperature, carefully remove aqueous phase to a new tube. Based on the amount of aqueous recovery from the previous step, add 1.25 times the volume of 100%ethanol to the aqueous phage in each tube and pipette to mix. Prepare fresh collection tubes containing a filter cartridge for each sample.Pipette approximately 675 microliters of the lysate-ethanol mixture onto the filter cartridge. Centrifuge briefly at 10, 000 times G to pass liquid through the filter. Discard the flow-through.Repeat the lysate-ethanol mixture adding to the filter and centrifugation until it has been completely used. Add 700 microliters of wash solution one from the kit to the filter cartridge. Centrifuge briefly to pull the solution through the filters.Discard the flow-through and keep the same filter cartridges and collection tubes. Add 500 microliters of wash solution two-three to each filter cartridge. After centrifuging again, discard the flow-through and repeat the wash step.To remove any residual liquid from the filter cartridges, spin them an additional 60 seconds. Transfer the cartridges to fresh collection tubes. Add 100 microliters of 95 degree Celsius preheated nuclease-free water to the center of each filter cartridge.Centrifuge the cartridges for approximately 20 to 30 seconds at the tabletop centrifuge at maximum speed. To preform hybridization with globin reduction oligos, first denature the RNA by adding each extracted sample into a 0.2 milliliter thin-walled nuclease-free reaction tube. Place the tubes in a thermal cycler at 70 degrees Celsius for two minutes.After that, immediately place the tubes on ice to obtain optimal RNA quality. While the tubes are cooling, prepare 400 microliters of 10X globin reduction oligo mix and 10X oligo hybridization buffer in a two milliliter tube. To make the hybridization mix, add six micrograms of RNA sample, two microliters of the 10X globin reduction oligo mix, one microliter of 10X oligo hybridization buffer, and nuclease free water to a final volume of 10 microliters to each 0.2 milliliter thin-walled, nuclease-free reaction tube.Place the tubes in the thermal cycler at 70 degrees Celsius for two minutes. After that, immediately place the tubes on ice. To perform RNase H digestion, first dilute 10X RNase H to one X RNase H with one X RNase H buffer.Prepare RNase H reaction mix by combining two microliters of 10X RNase buffer, one microliter of RNase inhibitor, and two microliters of one X RNase H, and five microliters of nuclease-free water to a total volume of 10 microliters. Add 10 microliters of the RNase H reaction mix to the globin reduction hybridization samples and mix thoroughly. Digest this reaction at 37 degrees Celsius for 10 minutes and then cool to four degrees Celsius.Stop digestion by adding one microliter of 0.5 molar EDTA to the tube, and transfer the entire content of the tube to a fresh 1.5 milliliter tube. Immediately after that, add 80 microliters of RNase-free water, 350 microliters of lysis buffer, then 250 microliters of 100%ethanol to each tube and mix well by pipetting. Transfer each 700 microliter sample to a separate elution filter cartridge placed into two milliliter collection tube to collect the flow-through.Centrifuge for 15 seconds at 8, 000 times G or greater and then discard the flow-through. Place the same elution filter cartridges into new two milliliter collection tubes. To wash the filter cartridge membranes, add 500 microliters of the mild washing buffer to the filter cartridges and centrifuge for 15 seconds at 8, 000 times G or greater.Discard the flow-through. Then, using the same collection tubes, add 500 microliters of 80%ethanol to the filter cartridges. Centrifuge for two minutes at 8, 000 times G and greater.Discard both the flow-through and collection tubes, and save the elution spin columns. Place each elution spin column into a new two milliliter collection tube. Centrifuge at full speed for five minutes with the open lid on filter cartridges to dry the spin column membranes and prevent ethanol carryover.After discarding the collection tubes, place each dried filter cartridge into a fresh 1.5 milliliter collection tube. Add 14 microliters of RNase-free water directly to the center of the filter cartridge membrane. To elute the RNA, centrifuge the tubes for 60 seconds at full speed and then continue with further RNA assessment and preparation.The globin-depleted sample can now be split and used to prepare the small, non-coding RNA libraries as well as the ribo-depleted mRNA and long, non-coding RNA libraries. After preparing expression libraries of the globin-depleted and ribo-depleted whole blood samples using this protocol, electrophoresis file run summary showed a globin-depleted library sample with RNA integrity numbers that ranged from 6.3 to 9.2. This proved to be an improvement compared to other studies that used globin depletion methods and were only able to achieve RIN numbers at or near six.Pre-globin-reduction and post-globin-reduction of a single porcine whole blood sample showed that 260 to 280 nanometer concentration ratios are at or above two. Using this protocol, chip-based electropherograms of globin-and ribo-depleted whole blood mRNA library samples prior to pooling and sequencing were obtained. For the mRNA libraries, the representative electropherogram has a peak at approximately 280 base pairs.For the small ncRNAs representative chip-based electropherograms contain a range of peaks from approximately 100 to 400 base pairs. Peaks at approximately 143 base pairs correspond to miRNAs, and peaks at approximately 153 base pairs correspond to piRNAs. While attempting this procedure, it's important to remember to ice tubes immediately where noted.Following this procedure, other methods such as next generation sequencing should be performed in order to answer additional questions such as differential expression, epigenetic changes, and their effect on biological pathways and processes. Don't forget that working with phenol-chloroform reagent is extremely hazardous and precautions such as working in a fume hood should be taken. Also, when handling whole blood or infectious organisms, work should be performed in a biological safety cabinet prior to an activation of the infectious organism.

identification-coding-non-coding-rna-classes-expressed-swine-whole

Research

JoVE Journal

Immunology and Infection

Depletion of Specific Cell Populations by Complement Depletion

The purification of immune cell populations is often required in order to study their unique functions. In particular, molecular approaches such as real-time PCR and microarray analysis require the isolation of cell populations with high purity. Commonly used purification strategies include fluorescent activated cell sorting (FACS), magnetic bead separation and complement depletion. Of the three strategies, complement depletion offers the advantages of being fast, inexpensive, gentle on the cells and a high cell yield. The complement system is composed of a large number of plasma proteins that when activated initiate a proteolytic cascade culminating in the formation of a membrane-attack complex that forms a pore on a cell surface resulting in cell death1. The classical pathway is activated by IgM and IgG antibodies and was first described as a mechanism for killing bacteria. With the generation of monoclonal antibodies (mAb), the complement cascade can be used to lyse any cell population in an antigen-specific manner. Depletion of cells by the complement cascade is achieved by the addition of complement fixing antigen-specific antibodies and rabbit complement to the starting cell population. The cells are incubated for one hour at 37&deg;C and the lysed cells are subsequently removed by two rounds of washing. MAb with a high efficiency for complement fixation typically deplete 95-100% of the targeted cell population. Depending on the purification strategy for the targeted cell population, complement depletion can be used for cell purification or for the enrichment of cell populations that then can be further purified by a subsequent method.

To effectively study the function of immune cell populations their purification is often required. Complement depletion is a fast and inexpensive technique for the isolation of immune cell populations with high purity.

The purification of immune cell populations is often required in order to study their unique functions.  In particular, molecular approaches such as ...

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Enumeration of Major Peripheral Blood Leukocyte Populations for Multicenter Clinical Trials Using a Whole Blood Phenotyping Assay

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many advantages for ease and standardization of immunological assessments, but detrimental effects of this process have been observed on some cell subsets, such as granulocytes, B cells, and dendritic cells 1-3. Assaying fresh leukocytes gives a more accurate picture of the in vivo state of the cells, but is often difficult to perform in the context of large clinical trials. Fresh cell assays are dependent upon volunteer commitments and timeframes and, if time-consuming, their application can be impractical due to the working hours required of laboratory personnel. In addition, when trials are conducted at multiple centers, laboratories with the resources and training necessary to perform the assays may not be located in sufficient proximity to clinical sites. To address these issues, we have developed an 11-color antibody staining panel that can be used with Trucount tubes (Becton Dickinson; San Jose, CA) to phenotype and enumerate the major leukocyte populations within the peripheral blood, yielding more robust cell-type specific information than assays such as a complete blood count (CBC) or assays with commercially-available panels designed for Trucount tubes that stain for only a few cell types. The staining procedure is simple, requires only 100 &mu;l of fresh whole blood, and takes approximately 45 minutes, making it feasible for standard blood-processing labs to perform. It is adapted from the BD Trucount tube technical data sheet (<a href="http://www.bdbiosciences.com/external_files/is/doc/tds/Package_Inserts_CE/live/web_enabled/23-3483-06_PI_Trucount tubes_CE_EN.pdf" target="_blank">version 8/2010</a>). The staining antibody cocktail can be prepared in advance in bulk at a central assay laboratory and shipped to the site processing labs. Stained tubes can be fixed and frozen for shipment to the central assay laboratory for multicolor flow cytometry analysis. The data generated from this staining panel can be used to track changes in leukocyte concentrations over time in relation to intervention and could easily be further developed to assess activation states of specific cell types of interest. In this report, we demonstrate the procedure used by blood-processing lab technicians to perform staining on fresh whole blood and the steps to analyze these stained samples at a central assay laboratory supporting a multicenter clinical trial. The video details the procedure as it is performed in the context of a clinical trial blood draw in the HIV Vaccine Trials Network (HVTN).

In this report, we demonstrate the staining and analysis steps of a phenotyping assay performed on fresh whole blood to enumerate major innate and adaptive leukocyte populations. We emphasize considerations for performing these procedures in the context of a multicenter clinical trial.

Cryopreservation of peripheral blood leukocytes is widely used to preserve cells for immune response evaluations in clinical trials and offers many ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of &#34;intrinsic&#34; patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native &#34;intrinsic pathogenic&#34; disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Here we describe a single-cell proteomic approach to evaluate immune phenotypic and functional (intracellular cytokine induction) alterations in peripheral whole blood samples, analyzed via mass cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies ...