Short Term Scientific Missions (STSMs)
Title: Regulation of innate immunity in pigs challenged with PRRSV by porcine
microarray analysis
Research memory
Background
Porcine reproductive and respiratory syndrome (PRRS) is an economically important
disease causing highly significant losses to the swine industry worldwide. Since the
early 1990s, vaccine therapy and management practices strategies have had a limited
impact on the spread of the disease. PRRS remains a challenge to the sustainability of
pig production (Mateu et al., 2008). The major concern of controlling PRRSV are the
highly genetic and, therefore, antigenic variability (Darwich et al., 2010, 2011; Gimeno
et al., 2010) and the dysregulation of the host response by the virus (Lemke et al., 2004,
Ait-Ali et al., 2011). New tools in genomics and transcriptomics will provide
information useful to gain insight into the genetic control of PRRS resistance or
tolerance, to identify loci implicated in between-host variation in resistance to PRRSV
infections and in disease outcomes in PRRS-affected herds.
Thus, the objective of this research collaboration is to screen differences in genes
involved in the regulation of innate and specific immune responses by microarray
(GeneChip Porcine Genome Array, Affymetrix UK, Ltd) (Ait-Ali et al., 2007, 2011),
between pigs that are well responders to a PRRSV vaccine (in terms of cellular and
humoral immunity) and pigs that are none or poor responders. Finally differences
among innate response regulatory genes (IFN-type I; MyD88; NF-kb; IRF3; IRF7; etc)
and regulation of antiviral Toll-like receptors (TLRs) will be analyzed.
Bibliography:
Ait-Ali et al (2011). Immunogenetics 63:437-448
Ait-Ali et al (2007). Viral Immunology 20: 105-118
Darwich et al. (2010). Virus Res.154(1-2):123-32
Darwich et al. (2011). Vet Mirobiol 150(1-2):49-62
Gimeno et al. (2011) Vet Research 42(1):9.
Lemke et al. (2004) J Immunol 172(3):1916-25
Mateu E and Díaz I. (2008). Vet J 177(3):345-51.
Material and methods
Animals:
Four week-old pigs, Landrace x Large White free of conventional pig pathogens
(PRRSV, TTV, PCV2, influenza, ADV, PPV, Mycoplasma hyopneumoniae). Pigs will
be vaccinated with a commercial PRRSV vaccine.
Groups:
1. Control (n=4): no vaccinated
2. Vaccinated (n=8): at time 0 are vaccinated with a commercial PRRSV vaccine. After
the results of IFN-gamma ELISPOTs and the humoral immuno responses, the
vaccinated pigs were classified in two different groups:
o vaccine well responders (n=4)
o vaccine bad responders (n=4)
Time points:
Two time points: At 3 weeks Post vaccination (PV) and 10 weeks PV
Samples:
Blood samples with heparin will be collected of all animals and PBMCs isolated by
Histopaque. For each animal, PBMCs (5x105 cells/well) will be cultured in medium
(mock, control), the PRRSV vaccine (moi 0.1, VAC), and phytohemagglutinin (10
μg/mL, PHA).
Total cells per eppendorf and stimulus: ranged from 2-6 x 106 cells resuspended in 1mL
of RNA later and preserved at -80ºC.
Extraction and quantification of RNA
The RNA extraction was done using the standardized protocol based on Trizol and
chloroform (1-bromo-3-Chloropropane) method. Depending on the final pellet, this was
resuspended in water MilliQ from15 to 50 ul.
The final RNA concentration was measured in the Nanodrop system (RNA
quantification). The method allows quantification of sRNA and the assessment of the
A260/280 ratio (see an example of the measurements):
Module:
Nucleic Acid
Path:
10 mm
Software:
3,3
Firmware:
USB2000 2.41.3 ND3
Sample ID
User ID
Date
ng/ul
A260
A280
Laila
13/08/2012
0
0
0
NaN
4 control t3
Laila
13/08/2012
70,7
1,767
0,952
1,86
4 pha t3
Laila
13/08/2012
209,46 5,236
2,699
1,94
4 vac t3
Laila
13/08/2012
74,86
1,01
1,85
1,871
The lectures of all examined animals are showed below:
a) At time 3wPV
PIG
REFERENCE
Sample ID
4 Control
4 PHA
4 VAC
5 Control
5 PHA
5 VAC
14 Control
14 PHA
14 VAC
16 Control
16 PHA
16 VAC
21 Control
21 PHA
21 VAC
24 Control
24 PHA
24 VAC
34 Control
34 PHA
34 VAC
38 Control
Total
volume
ul
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
15
RNA
quantification
ng/ul
81.43
187.95
144.27
127.53
151.96
115.19
80.13
396.42
179.9
195.1
191.33
172.83
172.86
331.32
263.75
125.96
275.42
194.87
187.47
203.48
225.47
117.2
260/280 Ratio
1.68
1.78
1.66
1.72
1.77
1.69
1.64
1.75
1.59
1.43
1.72
1.62
1.62
1.76
1.68
1.69
1.79
1.65
1.52
1.65
1.64
1.53
260/280
38 PHA
38 VAC
49 Control
49 PHA
49 VAC
61 Control
61 PHA
61 VAC
62 Control
62 PHA
62 VAC
63 Control
63 PHA
63 VAC
15
15
15
15
15
15
15
15
15
15
15
15
15
15
90.83
114.11
247.73
240.02
204.44
124.92
110.19
93.08
63.75
156.62
147.05
157.17
124.69
111.09
1.54
1.57
1.57
1.77
1.65
1.58
1.68
1.7
1.57
1.73
1.82
1.59
1.58
1.87
RNA
quantification
ng/ul
70.7
209.46
74.86
127.13
293.83
118.79
125.24
242.17
138.09
140.21
286.41
139.37
139.75
332.94
163.13
97.2
232.52
120.42
119.71
316.15
149.81
203.83
404.99
205.04
463.44
633.15
104.1
218.6
313.56
260/280 Ratio
at time 10wPV
PIG
REFERENCE
Sample ID
4 CONTROL T3
4 PHA T3
4 VAC T3
5 CONTROL T3
5 PHA T3
5 VAC T3
16 CONTROL T3
16 PHA T3
16 VAC T3
21 CONTORL T3
21 PHA T3
21 VAC T3
24 CONTRIOL T3
24 PHA T3
24 VAC T3
34 CONTROL T3
34 PHA T3
34 VAC T3
26 CONTROL T3
26 PHA T3
26 VAC T3
38 CONTROL T3
38 PHA T3
38 VAC T3
49 CONTROL T3
49 PHA T3
49 VAC T3
59 CONTROL T3
59 PHA T3
Total
volume
ul
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
1.86
1.94
1.85
1.92
1.93
1.87
1.82
1.93
1.92
1.82
1.95
1.87
1.89
1.93
1.87
1.82
1.91
1.78
1.95
2.01
1.97
1.96
1.99
1.96
1.65
1.76
1.63
1.78
1.59
59 VAC T3
61 CONTROL T3
61 PHA T3
61 VAC T3
63 CONTROL T3
63 PHA T3
63 VAC T3
50
50
50
50
50
50
50
398.02
583.8
620.36
622.27
212.31
131.39
451.15
1.94
1.94
1.63
1.59
1.9
1.97
1.89
Determination of the Quality of RNA
The quality and purity of the extracted RNA was determined using an Agilent 2100
Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies, Palo Alto, CA, USA) as
previously described (Ait-Ali et al, 2011).
This kit offers the ability to check RNA quality, including assignment of an RNA
Integrity Number (RIN). I used the Bioanalyzer to assess total RNA quantity, integrity
and ribosomal ratios for my samples. In addition, I used the RIN number as a standard
integrity measure that is independent of concentration or instrument. I routinely used the
RNA 6000 Nano Kit, which is designed to quantify total RNA from 25-500 ng/ul and
will provide a qualitative value when in the 5-500 ng/ul range.
An example of the assessment by the Nano kit is provided below:
Example of the RNA curves and RIN values displayed by the software. Samples of
PBMCs of different animals treated with the different stimulus:
Samples with a RIN value > 7 were suitable for the microarray analyses. The total RNA
of the selected samples was amplified and labeled using kits and instructions supplied
by Affymetrix (Affimetrix UK Ltd, High Wycombe, UK). The GeneChip Porcine
Genome Array (Affymetrix UK Ltd) comprises 23,937 probe sets that interrogate
approximately 23,256 transcripts from 20,201 Sus scrofa genes. The sequence
information for this array was selected from public data sources as described by Ait-Ali
et al. (2011).
More detailed information given by the kit:
Microarray data analysis, clustering, multidimensional scaling and heat map generation
will be performed by the University of Glasgow Bioinformatics unit using FunAlyse
software package that combines Robust Multichip Analysis, Rank Products and
Iterative Group Analysis methods.
The results of this work will be prepared in collaboration by both institutions (The
Roslin Institute and The Centre de Recerca en Sanitat Animal –CReSA-) in the next two
months, and a manuscript will be prepared for submitting in a peer-review journal.