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...

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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.

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