Name: Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood (Video) | JoVE Journal
Uploaded: 2018-11-28T14:30:00.000+00:00
Duration: 9 min 40 s
Description: Learn a protocol for isolating coding and non-coding RNA from swine whole blood using globin-reduced RNA-seq. This method enables researchers to study host-pathogen interactions and viral RNA expression.

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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.

<ol><li>Finotello, F., Di Camillo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measuring+differential+gene+expression+with+RNA-seq%3A+challenges+and+strategies+for+data+analysis%5BTitle%5D)+AND+%22Briefings+in+Functional+Genomics%22%5BJournal%5D)">Measuring differential gene expression with RNA-seq: challenges and strategies for data analysis.</a> Briefings in Functional Genomics. 14 (2), 130-142 (2015).</li>
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<li>Pollet, S. FR-AgEncode: a French pilot project to enrich the annotation of livestock genomes- RNA Extraction Protocol. , Available from: 
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<li>Fleming, D. S., Miller, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+small+non-coding+RNA+classes+expressed+in+swine+whole+blood+during+HP-PRRSV+infection%5BTitle%5D)+AND+%22Virology%22%5BJournal%5D)">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/pubmed?term=((Small+non-coding+RNA+expression+status+in+animals+faced+with+highly+pathogenic+porcine+reproductive+and+respiratory+syndrome+virus+%28HP-PRRSV%29%5BTitle%5D)+AND+%22Proceedings+of+the+World+Congress+on+Genetics+Applied+to+Livestock+Production%22%5BJournal%5D)">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), Species - Porcine 2 (2018).</li>
<li>Bivens Nathan, J., Zhou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-Seq+Library+Construction+Methods+for+Transcriptome+Analysis%5BTitle%5D)+AND+%22Current+Protocols+in+Plant+Biology%22%5BJournal%5D)">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/pubmed?term=((A+comparison+between+ribo-minus+RNA-sequencing+and+polyA-selected+RNA-sequencing%5BTitle%5D)+AND+%22Genomics%22%5BJournal%5D)">A comparison between ribo-minus RNA-sequencing and polyA-selected RNA-sequencing.</a> Genomics. 96 (5), 259-265 (2010).</li>
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<li>Choi, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Increasing+gene+discovery+and+coverage+using+RNA-seq+of+globin+RNA+reduced+porcine+blood+samples%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Increasing gene discovery and coverage using RNA-seq of globin RNA reduced porcine blood samples.</a> BMC Genomics. 15, 954(2014).</li>
<li>Wu, K., Miyada, G., Martin, J., Finkelstein, D. Globin reduction protocol: A method for processing whole blood RNA samples for improved array results. , Available from: 
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<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: 
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<li>Shin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Variation+in+RNA-Seq+transcriptome+profiles+of+peripheral+whole+blood+from+healthy+individuals+with+and+without+globin+depletion%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Uncovering+the+complexity+of+transcriptomes+with+RNA-Seq%5BTitle%5D)+AND+%22Journal+of+Biomedicine+and+Biotechnology%22%5BJournal%5D)">Uncovering the complexity of transcriptomes with RNA-Seq.</a> Journal of Biomedicine and Biotechnology. 2010, 853916(2010).</li>
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The authors have nothing to disclose.
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

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.

<table><tbody><tr><td>PAXgene Tubes</td><td>PreAnalytix</td><td>762165</td><td></td></tr><tr><td>Molecular Biology Grade Water</td><td>ThermoFisher</td><td>10977-015</td><td></td></tr><tr><td>mirVana miRNA Isolation Kit</td><td>ThermoFisher</td><td>AM1560</td><td></td></tr><tr><td>Rneasy MinElute Clean Up Kit</td><td>QIAGEN</td><td>74204</td><td></td></tr><tr><td>100% Ethanol</td><td>Decon Labs, Inc.</td><td>2716</td><td></td></tr><tr><td>0.2 mL thin-walled tubes</td><td>ThermoFisher</td><td>98010540</td><td></td></tr><tr><td>1.5 mL RNase/DNase - free tubes</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Veriti 96-well Thermocycler</td><td>ThermoFisher</td><td>4375786R</td><td></td></tr><tr><td>Globin Reduction Oligo (&amp;alpha; 1)</td><td>Any supplier</td><td></td><td>Sequence GAT CTC CGA GGC TCC AGC TTA ACG GT</td></tr><tr><td>Globin Reduction Oligo (&amp;alpha; 2)</td><td>Any supplier</td><td></td><td>Sequence TCA ACG ATC AGG AGG TCA GGG TGC AA</td></tr><tr><td>Globin Reduction Oligo (&amp;beta; 1)</td><td>Any supplier</td><td></td><td>Sequence AGG GGA ACT TAG TGG TAC TTG TGG GT</td></tr><tr><td>Globin Reduction Oligo (&amp;beta; 2)</td><td>Any supplier</td><td></td><td>Sequence GGT TCA GAG GAA AAA GGG CTC CTC CT</td></tr><tr><td>10X Oligo Hybridization Buffer</td><td></td><td></td><td></td></tr><tr><td>-Tris-HCl, pH 7.6</td><td>Fisher Scientific</td><td>BP1757-100</td><td></td></tr><tr><td>-KCl</td><td>Millipore Sigma</td><td>60142-100ML-F</td><td></td></tr><tr><td>10X RNase H Buffer</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;-Tris-HCl, pH 7.6</td><td>Fisher Scientific</td><td>BP1757-100</td><td></td></tr><tr><td>&amp;nbsp;-DTT</td><td>ThermoFisher</td><td>Y00147</td><td></td></tr><tr><td>&amp;nbsp;-MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Promega</td><td>A351B</td><td></td></tr><tr><td>&amp;nbsp;-Molecular Biology Grade Water</td><td>ThermoFisher</td><td>10977-015</td><td></td></tr><tr><td>RNase H</td><td>ThermoFisher</td><td>AM2292</td><td></td></tr><tr><td>SUPERase-IN</td><td>ThermoFisher</td><td>AM2694</td><td>Rnase inhibitor</td></tr><tr><td>EDTA</td><td>Millipore Sigma</td><td>E7889</td><td></td></tr><tr><td>Microcentrifuge</td><td>Any supplier</td><td></td><td></td></tr><tr><td>&amp;nbsp;2100 Electrophoresis BioAnalyzer Instrument</td><td>Agilent Technologies</td><td>G2938C</td><td></td></tr><tr><td>Agilent RNA 6000 Nano Kit</td><td>Agilent Technologies</td><td>5067-1511</td><td></td></tr><tr><td>Agilent High Sensitivity DNA&amp;nbsp; Kit</td><td>Agilent Technologies</td><td>5067-4626</td><td></td></tr><tr><td>TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero&amp;nbsp;</td><td>Illumina</td><td>RS-122-2201</td><td>mRNA kit; Human/Mouse/Rat Set A&amp;nbsp; (48 samples, 12 indexes)</td></tr><tr><td>TruSeq Stranded Total RNA Sample Preparation Guide</td><td>Illumina</td><td></td><td>Available on-line</td></tr><tr><td>RNAClean XP Beads</td><td>BeckmanCoulter</td><td>A63987</td><td></td></tr><tr><td>AMPure XP Beads</td><td>BeckmanCoulter</td><td>A63880</td><td></td></tr><tr><td>MicroAmp Optical 8-tube Strip</td><td>ThermoFisher</td><td>N8010580</td><td>0.2 ml thin-walled tubes</td></tr><tr><td>MicroAmp Optical 8-tube Strip Cap</td><td>ThermoFisher</td><td>N801-0535</td><td></td></tr><tr><td>RNase/DNase - free reagent reservoirs</td><td>Any supplier</td><td></td><td></td></tr><tr><td>SuperScript II Reverse Transcriptase</td><td>ThermoFisher</td><td>18064-014</td><td></td></tr><tr><td>MicroAmp Optical 96 well plates</td><td>ThermoFisher</td><td>N8010560</td><td>These were used in place of .3mL plates as needed</td></tr><tr><td>MicroAmp Optical adhesive film</td><td>ThermoFisher</td><td>4311971</td><td></td></tr><tr><td>NEBNext Multiplex Small RNA Library Prep Set for Illumina&amp;reg; (Set 1)&amp;nbsp;</td><td>New England Biolabs</td><td>E73005</td><td>small RNA kit</td></tr><tr><td>NEBNext Multiplex Small RNA Library Prep Set for Illumina&amp;reg; (Set 2)&amp;nbsp;</td><td>New England Biolabs</td><td>E75805</td><td>small RNA kit</td></tr><tr><td>QIAQuick PCR Purification Kit</td><td>QIAGEN</td><td>28104</td><td></td></tr><tr><td>96S Super Magnet Plate</td><td>ALPAQUA</td><td>A001322</td><td></td></tr></tbody></table>

identification of coding and non-coding rna classes expressed in swine whole blood

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...

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 ...

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Learn a protocol for isolating coding and non-coding RNA from swine whole blood using globin-reduced RNA-seq. This method enables researchers to study host-pathogen interactions and viral RNA expression.

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

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