ONLINE RESOURCE 1

Negative Energy Balance Alters Gene Expression Profiles of Bovine Blood Neutrophils During Intramammary Mastitis Challenge With Streptococcus uberis.

Kasey M. Moyes†*, James K. Drackley†, Dawn E. Morin‡, and Juan J. Loor†#

†Department of Animal Sciences, and

‡College of Veterinary Medicine, University of Illinois, Urbana, 61801

*Current address: Faculty of Agricultural Sciences, Århus University, Blichers Allé, P.O. Box 50, Tjele, Denmark 8830

#Corresponding author: Dr. Juan J. Loor, 1207 W. Gregory Dr., Urbana, IL 61801, Phone: (217) 244-5957, Fax: (217) 333-8286

RNA isolation. Total RNA was isolated from blood PMN using TRIzol®Reagent (Invitrogen, Carlsbad, CA). Isolated neutrophils (see main manuscript for details) were homogenized in TRIzol reagent with 1 µL linear acrylamide (Ambion, Inc., Austin, TX) using a Tissue-Tearor (BioSpec Products, Inc.) homogenizer at maximum speed. Upon centrifugation, total RNA wasseparated with chloroform followed by acid phenol:chloroform (Ambion, Inc., Austin, TX) to remove DNA. Total RNA was then precipitated with isopropanol and the RNA pellet was cleaned with 75% ethanol prior to reconstitution in RNA storage buffer (Ambion, Inc., Austin, TX) and storage at -80°C. Total RNA was cleaned using RNeasy mini-kit columns and residual DNA removed using the RNase-Free DNase Set (Qiagen, Valencia, CA) prior to microarray or qPCR. RNA quality was assessed using a 2100 Bioanalyzer (Agilent Tenchnologies). Purity of RNA were assessed by ration of optical density OD260mm(oligonucleotides assorbance) /OD280mm (protein assorbance) using NanoDrop ND-1000 (NanoDrop Technologies, Rockland, DE). The ratio of the sample was > 1.7.

Primer design and testing for qPCR. Primers were designed as previously described (Bionaz and Loor 2007). Briefly, Primer Express software version 3.0 (Applied Biosystems, Foster City, CA) was used with defaults features except min amplicon length to 100. Primers were aligned using publicly available databases including BLASTN at NCBI and UCSC’s Cow (Bos taurus) Genome Browser Gateway ( Primers were first tested using the SYBR Green protocol as describe below without the dissociation protocol. Five L of the PCR products were run on a 2% agarose gel (Invitrogen, Carlsbad, CA) stained with ethidium bromide to assess presence of the product to a expected size and presence of primer-dimer, the rest was purified using Qiaquick PCR purification kit (Qiagen, Valencia, CA) and sent to Core DNA Sequencing Facility of the Roy J. Carver Biotechnology Center at the University of Illinois, Urbana, for sequencing. Online Resource Table 1below shows the list of all primers designed and used for qPCR analysis in this study. All sequences were confirmed using NCBI and UCSC’s cow Genome Browser Gateway and are shown in Online Resource Tables 2 and 3 below.

Identification of internal control genes. Detailed description of the selection criteria of internal control genes (ICG) for qPCR analysis was provided by Piantoni et al. (2008). Internal control genes were identified using a specific bovine 13K oligoarray previously described (Everts et al. 2005; Loor et al. 2005; PMN microarray data unpublished). This platform is publicly accessible in the NationalCenter for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (GPL8776). The microarray data files used to identify ICG in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO; and are accessible through GEO series accession number [GSE15364].

Briefly, GeneSpring GX software Version 3 (Agilent Technologies,Santa Clara, CA)to identify PMN gene expression stability among all samples, i.e. TRT (NEB and PEB). Genes with a constant normalized expression ratio (~1.0; sample/reference) and the greatest presence on microarrays (i.e., 20 out of 20 arrays, 10 cows, and >100 relative fluroscence unit) were further used for ICG co-regulation evaluation using Ingenuity Pathway Analysis (IPA®; Redwood City, CA). Co-regulation is described as the presence of common upstream regulator factors or direct regulation of transcript expression between gene products. Genes without known co-regulation from the selected genes or transcription factors were further used for ICG evaluation through geNorm (Vandesompele et al. 2002). These included GOLGA5, GTP, OSBPL2, and SMUG1. Next, gene stability of expression ratios among potential ICG was evaluated using geNorm software following the pairwise comparison method (Vandesompele et al. 2002; Online Resource Figure 1 Asee below). geNorm determines both the optimal number of ICG (Online Resource Figure 1 B see below) to obtain a reliable normalization factor (NF) and calculates the NF. The optimal number of ICG were determined by assessment of the pairwise variation V (Vn/n+1) between the normalization factors NFn and NFn+1. Due to the very high stability of NF using GOLGA5, OSBPL2, and SMUG1, these 3 genes were used for calculation of NF. The V of NF using GOLGA5, OSBPL2, and SMUG1 (V < 0.004) proved to be even more stable than those used initially.

qPCR. For cDNA synthesis, 100 ng of purified (described above) RNA sample was combined with 1 µg dT18 (Operon Biotechnologies Inc., Germantown, MD), 1 µL 10 mM dNTP mix (Invitrogen, Carlsbad, CA), 1 µL Random Primers (Invitrogen, Carlsbad, CA), and 7 µL DNase/RNase free water. The mixture was incubated for 5 min at 65°C using an Eppendorf Mastercycler Gradient and then placed on ice for 3 min. A total of 9 µL of Master Mix was added, consisting of 4.0 µL 5X First-Strand Buffer (Invitrogen, Carlsbad, CA), 1 µL 0.1 M DTT, 0.25 µL of SuperScriptTMIII reverse transcription (Invitrogen, Carlsbad, CA), 0.25 µL of RNase Inhibitor (Promega, Madison, WI), and 3.5 µL of DNase/RNase free water. The mixture was incubated for 5 min at 25°C followed by 60 min at 50°C and then for 15 min at 70°C. The cDNA was then diluted 1:4 with DNase/RNase free water. A combination of 4 µL of diluted cDNA with 6 µL of 1×SYBR Green master mix (Applied Biosystems, Foster City, CA), 0.4 µLeach of 10 µM forward and reverse primers, and 0.2 µL of DNase/RNase free water were added to each well of a MicroAmp Optical 384-Well Reaction Plate (Applied Biosystems). All samplesand a 6-point standard curve (see below) wereanalyzed in triplicate. The reactions were performed in an ABI Prism 7900 HT SDS instrument (Applied Biosystems) under the following conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 s at 95°C, and 1 min at 60°C. During the last cycle (95°C for 15 s, 65°C for 15 s), a dissociation protocol was used to specify the presence of a single PCR product. The standard curve was generated by pooling cDNA from all 8 of 10 experimental samples (8 out of 10 cows). Two of the 10 samples were unusable for qPCR analysis due to the low amount of RNA isolated. A 6-point standard curve was generated via serial dilution of the pooled standard using DNase/RNase free water to obtain a 1:5 (v:v) or 20% dilution per point. Online Resource Table 4below shows the slope and coefficient of determination of the standard curve, efficiency, and median cycle threshold (Ct) of the measured transcripts from qPCR SDS documents generated from the ABI Prism 7900 HT SDS instrument.

Data mining. Data were mined by means of Ingenuity Pathways Analysis (IPA, Ingenuity® Systems, The 12 differentially expressed genes (DEG) with qPCR P ≤ 0.05 were uploaded into IPA with associated annotation and least squares means. Ingenuity Pathways Knowledge Base was used as a reference set for statistical analysis of enriched networks among DEG. Each gene was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base. The 12DEG were run without fold-change cut-off.

Online Resource Table 1. GenBank accession number, hybridization position, sequence, amplicon size, and source of primers for Bos taurus used to analyze gene expression by qPCR.
Accession # / Gene / Primer / Primers (5’- 3’) / Amplicon Size (bp) / Source
DQ059505 / ABCA1 / F.972 / CGGCGGCTTCTCTTGTATAGC / 101 / This manuscript
R.1072 / TTCAAGCGTGAGCTGAAACG
BC118389 / ALOX5AP / F.114 / ACAAGGTGGAGCACGAAAGC / 100 / This manuscript
R.213 / ACACAGTTCTGGTTGGCAGTGT
BC142290 / CPNE3 / F.161 / GAGAAGGAGCCAGCTCAAGAGA / 100 / This manuscript
R.260 / ACTTTGACCCTATGTCTTTATCCAGAA
BC140485 / GOLGA5 / F.1370 / GAGCTACAGCAGCAAGTCAAAGTG / 103 / This manuscript
R.1472 / CTTTAGACTGGAGTATTCGAGTAGCTTTT
BC114669 / GRB7 / F.1064 / CCCACAGACTTCGGCTTCTG / 141 / This manuscript
R.1204 / CAGCTGTACCCCAAACTTGAAGA
AK074976 / GTP / F.696 / CTTGGAATCCGAGGAGCCA / 100 / Bionaz and Loor 2007
R.796 / CCTGGGATCACCAGAGCTGT
NM_001040498 / HLA-A / F.1017 / ACGCAGACCTTCACCACATACTC / 100 / This manuscript
R.1116 / GAAGCATCACTCAGTCCCCATAA
BT021059 / HLA-DRA / F.594 / AAGTTCCACTACCTCCCCTTCCT / 110 / This manuscript
R.703 / GGAGCTTCATACTCCCAGTGCTT
M37211 / IL1B / F.30 / ATTCTCTCCAGCCAACCTTCATT / 100 / This manuscript
R.129 / TTCTCGTCACTGTAGTAAGCCATCA
BC112665 / IL1R2 / F.980 / AGCGCCAGGCATACTCAGAA / 90 / This manuscript
R.1069 / AATCCGTGTGCAAATCCTCTCT
BC134577 / IL1RN / F.308 / GCTCAAGTTAGAGGCCGTGAA / 118 / This manuscript
R.425 / GCAGGCAGCTGACTCAAAGC
BC123577 / IL6 / F.290 / TGAGTGTGAAAGCAGCAAGGA / 101 / Loor et al. 2007
R.529 / GCATCTTCGTTGTCATGTAGGTTT
EU276073 / IL8 / F.102 / TGTGAAGCTGCAGTTCTGTCAA / 130 / This manuscript
R.231 / TTTCACAGTGTGGCCCACTCT
DQ319075 / IRAK-1 / F.950 / CCTCAGCGACTGGACATCCT / 103 / This manuscript
R.1052 / GGACGTTGGAACTCTTGACATCT
DN283323 / NFKB1A / F.391 / GGGAGACCTGGCCTTCCTC / 101 / This manuscript
R.491 / CCAGAAGTGCCTCAGCGATT
BT021611 / OSBPL2 / F.198 / TGCCGTCACAGGCTTTGAC / 100 / This manuscript
R.297 / CCATTACTTGCTGGTGTCCACAT
BC140620 / PRKCB1 / F.1091 / ACAGAAATTCGAGAGAGCCAAGA / 100 / This manuscript
R.1190 / GTCCCTGTTGCCGTTGTTGT
NM_174182 / SELL / F.588 / CTCTGCTACACAGCTTCTTGTAAACC / 104 / This manuscript
R.691 / CCGTAGTACCCCAAATCACAGTT
BC148122 / SMUG1 / F.258 / CAGCTACGTGACCCGCTACTG / 117 / This manuscript
R.374 / CGGACTACACTCACTTCACCAAAG
BC102432 / SOD1 / F.256 / GGCTGTACCAGTGCAGGTCC / 101 / This manuscript
R.356 / GCTGTCACATTGCCCAGGT
TC263657 / SREBF1 / F.143 / CCAGCTGACAGCTCCATTGA / 67 / Loor et al. 2005
R.209 / TGCGCGCCACAAGGA
BC104530 / THY1 / F.332 / GGTCCTCTACCTGTCCAACTTCA / 127 / This manuscript
R.458 / CCTGACCAGTTTATCTCTCAACACA
AF310951 / TLR2 / F.88 / TGGGTGGAGAACCTCATGGT / 100 / This manuscript
R.187 / CGATAATCCACTTGCCAGGAA
AF310952 / TLR4 / F.85 / TGCGTACAGGTTGTTCCTAACATT / 110 / This manuscript
R.194 / TAGTTAAAGCTCAGGTCCAGCATCT
AF348421 / TNF / F.174 / CCAGAGGGAAGAGCAGTCCC / 114 / This manuscript
R.287 / TCGGCTACAACGTGGGCTAC
Online Resource Table 2. Sequencing results obtained from qPCR product of Bos taurus specific primers.
Gene / Sequence
ABCA1 / GCGACGCATGAAGGACATACGCAAAGTTTTGAAGACGTTATACCAGATCGAGGCGTTTCAGCTCACGCATTGAAA
ALOX5AP / CGGGACTCCAGAGGACTGGGACGCTGGCCTTTGAGCGGGTCTACACTGCCAACCAGAACTGTGATAATTTT
CPNE3 / GGTGGAGCTGAATATTTCCTGTGACAATCTTCTGGATAAAGACATAGGGTCAAAGTAATTT
GOLGA5 / GTGGACATCCTCATGAGCAAGAATTAGCTGACTACAAGGGA
HLA-A / TCTCTCACGGTTCCTAAAGTGTGAGACTACCTGCCTTATGGGGACTGAGTGATGCCTTCCAA
HLA-DRA / GCAGGAGATGTCTATGACTGCAAGGTGGAGCACTTGGGTTTGAATGAGCCTCTTCTCAAGCACTGGGAGTATGAAGCTCCAAA
IL1B / ACAGCCATGGCACCGTACCTGAACCCATCAACGAAATGATCGGCTTACGTCACAGTGGACAGAGCACAATAGCACCCCC
IL1R2 / AAGGTGCCACTGATTTTTGATCCTGTCAGAAGAGAGGATTTTGGCCACCACCGGGATTAA
IL1RN / CAGTCAGTACGAACAGGGAGCAGGACAAGCGCTTTGCCTTCATCCGCTTCGACAACGGGCCCACCACCAGCTTTGAGTCAGCTGCCTGCAA
IL8 / GGATACTGACTTCGATGCCAATGCATAAAAACACATTCCACACCTTTCCACCCCAAATTTATCAAAGAATTGAGAGTTATTGAGAGATGGGCCACACTGTGAAAA
IRAK-1 / GCTACGGGCATCAGTTCTTACATCAAGATAGCCCCAGCCTCATCCATGGAGATGTCAAGAGTTCCAACGTCCAA
OSBPL2 / GCATGCACTGGGAATTTTCAGAGGCGAATCAGAGACTCACTGGCGTGATTCATGTGGACACCAGCAAGTAATGGATTTTT
PRCKB1 / CTGTCGTGCCAGACTCCTGAAGAAAAGACGACCAACACCATCTCCAAATTTGACAACAACGGCAACAGGGACAT
SELL / ACGGGCCATGGACAATGTGTGGAAGTCATCAATAATTACACCTGCGTGCCCGTGATTTGGGGGGACCTCCGGAA
SMUG1 / CGTCTCGTTCGATGGTCGAATGAATGCCTACCGGAACCCTTTGGCATGGCCCAGACGGGGGTGCCCTTTGGTGAATTTGAGTGTAGTCCGA
TLR2 / AGCATGGACTCACCTCCTTTAGCTGTGTCTTCATAAGCGAGACTTCATTCCTGGCAAGTGGATTATCGA
TLR4 / AGACTGGTCAGGAGCTGATCTCTACAAATCCCCGACAACATCCCCATATCAACCAAGATGCTGGACCTGAGCTTTAACTAAG
TNF / CTGGTTCAACACTCAGGTCCTCTTCTCAAGCCTCAAGTAACAAGCCGGTAGCCCACGCTTGTAGCCGAAA
Online Resource Table 3. Sequencing results of genes using BLASTN ( from NCBI against nucleotide collection with total score.
Accession # / Name / Best hit in NCBI / Score
DQ059505 / ABCA1 / Bos taurus ATP-binding cassette sub-family A member 1 / 111
NM_001076293 / ALOX5AP / Bos taurus arachidonate 5-lipoxygenase-activating protein mRNA, complete cds / 100
BC142290 / CPNE3 / Bos taurus copine III (CPNE3), mRNA / 102
BC140485 / GOLGA5 / Bos taurus golgi autoantigen, golgin subfamily a, 5 (GOLGA5), mrna / 46.1
NM_001040498 / HLA-A / Bos taurus MHC Class I JSP.1, mRNA, complete cds / 91.5
BT021059 / HLA-DRA / Bos taurus major histocompatibility complex, class II, DR alpha, mRNA, complete cds / 136
M37211 / IL1B / Bovine mRNA for interleukin-1 beta / 73.4
BC112665 / IL1R2 / Bos taurus interleukin 1 receptor, type II, mRNA, complete cds / 69.8
BC134577 / IL1RN / Bos taurus interleukin 1 receptor antagonist, mRNA, complete cds / 143
EU276073 / IL8 / Bos taurus interleukin 8 (IL8) mRNA, complete cds / 167
DQ319075 / IRAK-1 / Bos taurus interleukin-1 receptor-associated kinase 1 (IRAK1) mRNA, complete cds / 114
BT021611 / OSBPL2 / Bos taurus oxysterol binding protein-like 2 (OSBPL2), mRNA, complete cds / 118
BC140620 / PRKCB1 / Bos taurus protein kinase C, beta, mRNA, complete cds / 113
NM_174182 / SELL / Bos taurus selectin L (lymphocyte adhesion molecule 1) (SELL), mRNA / 92.5
BC148122 / SMUG1 / Bos taurus single-strand-selective monofunctional uracil-DNA glycosylase 1, mRNA (cDNA clone MGC:148572 IMAGE:8358645), complete cds / 87.8
EU746459 / TLR2 / Bos taurus isolate 74 toll-like receptor 2 (TLR2) gene, complete cds / 91.5
DQ839567 / TLR4 / Bos taurus toll-like receptor 4 (TLR4) gene, complete cds / 114
EU276079 / TNF / Bos taurus tumor necrosis factor alpha (TNFa) mRNA, complete cds / 109
Online ResourceTable 4. Slope1 and coefficient of determination of the standard curve (R2), efficiency (E)2, and median cycle threshold (Ct) of the measured transcripts.
Gene Symbol / Slope / R2 / E / Median Ct
ABCA1 / -3.07 / 0.997 / 2.12 / 22.1
ALOX5AP / -2.95 / 0.977 / 2.18 / 26.1
CPNE3 / -3.25 / 0.998 / 2.01 / 23.2
GOLGA5 / -3.29 / 0.998 / 2.01 / 23.5
GRB7 / -3.31 / 0.991 / 2.01 / 25.0
GTP / -3.49 / 0.972 / 1.93 / 23.4
HLA-A / -3.04 / 0.992 / 2.13 / 21.3
HLA-DRA / -3.31 / 0.996 / 2.01 / 20.6
IL1B / -3.11 / 0.995 / 2.10 / 23.9
IL1R2 / -3.46 / 0.997 / 1.94 / 27.1
IL1RN / -3.50 / 0.996 / 1.93 / 22.3
IL6 / -2.62 / 0.940 / 2.41 / 29.5
IL8 / -3.17 / 0.969 / 2.07 / 27.1
IRAK-1 / -3.42 / 0.994 / 1.96 / 23.0
NFKB1A / -3.41 / 0.992 / 1.97 / 21.5
OSBPL2 / -3.10 / 0.996 / 2.10 / 20.7
PRCKB1 / -3.06 / 0.991 / 2.12 / 25.9
SELL / -3.49 / 0.984 / 1.94 / 25.5
SMUG1 / -3.43 / 0.995 / 1.96 / 23.1
SOD1 / -3.04 / 0.997 / 2.13 / 23.5
SREBF1 / -3.13 / 0.998 / 2.00 / 21.6
THY1 / -3.31 / 0.994 / 2.00 / 22.8
TLR2 / -3.32 / 0.986 / 2.00 / 22.6
TLR4 / -3.63 / 0.990 / 1.87 / 25.0
TNF / -3.17 / 0.977 / 2.07 / 25.0
1Slope generated by 6-point standard curve.
2Efficiency = 10(-1/slope).

Online ResourceFigure 1. Stability (M) of gene expression ratios of potential ICG using geNorm software (A). geNorm analysis of optimal number of ICG for qPCR normalization for a subset of genes differentially expressed on microarray (B).