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

Summary

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

Abstract

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 size selection, to allow capture of more RNA species.

Introduction

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 individuals^1,2,3,4. The sequencing take place on a whole genome basis and allows a researcher to c....

Protocol

Animal protocols were approved by the National Animal Disease Center (USDA-ARS-NADC) Animal Care and Use Committee.

1. Collection of Swine Blood Samples

1. Collect blood samples into RNA tubes. Collect ~2.5 mL or more if larger collection tubes are available.

2. Processing of Swine Blood Samples

1. Centrifuge the blood tubes at 5,020 x g for 10 min at room temperature (15-25 °C). If processing frozen samples incubate tube at room temperature for a minimum of 2 h prior to centrifugation.
2. Remove supernatant and add 8 mL of RNase-free water to the pel....

Results

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
PAXgene Tubes	PreAnalytix	762165
Molecular Biology Grade Water	ThermoFisher	10977-015
mirVana miRNA Isolation Kit	ThermoFisher	AM1560
Rneasy MinElute Clean Up Kit	QIAGEN	74204
100% Ethanol	Decon Labs, Inc.	2716
0.2 mL thin-walled tubes	ThermoFisher	98010540
1.5 mL RNase/DNase - free tubes	Any supplier
Veriti 96-well Thermocycler	ThermoFisher	4375786R
Globin Reduction Oligo (α 1)	Any supplier		Sequence GAT CTC CGA GGC TCC AGC TTA ACG GT
Globin Reduction Oligo (α 2)	Any supplier		Sequence TCA ACG ATC AGG AGG TCA GGG TGC AA
Globin Reduction Oligo (β 1)	Any supplier		Sequence AGG GGA ACT TAG TGG TAC TTG TGG GT
Globin Reduction Oligo (β 2)	Any supplier		Sequence GGT TCA GAG GAA AAA GGG CTC CTC CT
10X Oligo Hybridization Buffer
-Tris-HCl, pH 7.6	Fisher Scientific	BP1757-100
-KCl	Millipore Sigma	60142-100ML-F
10X RNase H Buffer
-Tris-HCl, pH 7.6	Fisher Scientific	BP1757-100
-DTT	ThermoFisher	Y00147
-MgCl2	Promega	A351B
-Molecular Biology Grade Water	ThermoFisher	10977-015
RNase H	ThermoFisher	AM2292
SUPERase-IN	ThermoFisher	AM2694	Rnase inhibitor
EDTA	Millipore Sigma	E7889
Microcentrifuge	Any supplier
2100 Electrophoresis BioAnalyzer Instrument	Agilent Technologies	G2938C
Agilent RNA 6000 Nano Kit	Agilent Technologies	5067-1511
Agilent High Sensitivity DNA  Kit	Agilent Technologies	5067-4626
TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero	Illumina	RS-122-2201	mRNA kit; Human/Mouse/Rat Set A  (48 samples, 12 indexes)
TruSeq Stranded Total RNA Sample Preparation Guide	Illumina		Available on-line
RNAClean XP Beads	BeckmanCoulter	A63987
AMPure XP Beads	BeckmanCoulter	A63880
MicroAmp Optical 8-tube Strip	ThermoFisher	N8010580	0.2 ml thin-walled tubes
MicroAmp Optical 8-tube Strip Cap	ThermoFisher	N801-0535
RNase/DNase - free reagent reservoirs	Any supplier
SuperScript II Reverse Transcriptase	ThermoFisher	18064-014
MicroAmp Optical 96 well plates	ThermoFisher	N8010560	These were used in place of .3mL plates as needed
MicroAmp Optical adhesive film	ThermoFisher	4311971
NEBNext Multiplex Small RNA Library Prep Set for Illumina® (Set 1)	New England Biolabs	E73005	small RNA kit
NEBNext Multiplex Small RNA Library Prep Set for Illumina® (Set 2)	New England Biolabs	E75805	small RNA kit
QIAQuick PCR Purification Kit	QIAGEN	28104
96S Super Magnet Plate	ALPAQUA	A001322