Advances in Bioscience and Biotechnology, 2013, 4, 6-14 ABB
http://dx.doi.org/10.4236/abb.2013.410A3002 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Reference gene selection for quantitative PCR studies in
bovine neutrophils
William R. Vorachek1, Gerd Bobe2, Jean A. Hall1
1Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, USA
2Department of Animal and Rangeland Sciences, College of Agricultural Sciences, Linus Pauling Institute, Oregon State University,
Corvallis, USA
Received 30 June 2013; revised 31 July 2013; accepted 2 August 2013
Copyright © 2013 William R. Vorachek et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Reference genes are essential for studying mRNA ex-
pression with quantitative PCR (qPCR). We investi-
gated 11 candidate whole-blood neutrophil reference
genes (ACTB, B2M, G6PD, GAPDH, GYPC, HPRT,
PGK1, RPL19, SDHA, TFRC, and YWHAZ) for beef
calves, both males and females, with or without sele-
nium supplementation. Initial screening was based on
gene expression level (<28 Cq cycles), variability (SD
< 1.5 Cq cycles), excluded GYPC and TFRC from
further analysis. Expression stability of the remaining
genes was evaluated using four software programs:
geNorm, NormFinder, BestKeeper, and the compara-
tive delta Cq method. The neutrophil reference genes,
YWHAZ, PGK1, and RPL19, consistently ranked
among the top four most stable genes under these ex-
perimental conditions. The commonly used reference
genes, ACTB and HPRT, were not reliable, under-
scoring the need to validate neutrophil reference genes
under different experimental conditions. Multiple re-
ference genes rather than a single gene may provide
more robust and reliable results. The best pair of re-
ference genes in whole-blood neutrophils from beef
calves overall was PGK1|YWHAZ.
Keywords: Blood Neutrophils; Bos taurus; qPCR;
Reference Genes; Selenium Treatment
1. INTRODUCTION
Quantitative PCR (qPCR) is one of the most common
techniques used for evaluating gene expression [1]. To
adjust for differences in total RNA concentrations, data
are normalized against RNA levels of internal control
genes, also called reference genes or housekeeping genes.
Reference genes are assumed to be constitutively ex-
pressed in the tissue or cell type of interest at concentra-
tions similar to genes of interest; however, their RNA
levels may vary depending on tissue or cell type ana-
lyzed as well as experimental conditions [2-5]. Therefore,
it is essential to evaluate reference genes in the same
type of tissue and under similar experimental conditions
before using them to adjust RNA levels for genes of in-
terest.
Our interest is in studying the effects of supranutri-
tional selenium (Se) supplementation on immune func-
tion in ruminants. We previously reported in sheep that
supranutritional Se-yeast supplementation alters whole-
blood (WB) neutrophil gene expression profiles [6].
Neutrophils are the most numerous and important cellu-
lar component of innate immunity. They serve as the
body’s first line of defense against invading microorgan-
isms. Neutrophils use two general mechanisms to kill mi-
croorganisms: extracellular killing via neutrophil extra-
cellular traps [7-9], or intracellular killing via phagocy-
tosis followed by the secretion of antimicrobial proteins,
proteolytic enzymes, and reactive oxygen species when
membrane-bound granules fuse with phagocytic vesicles.
We and others have reported on potential reference genes
in human [10,11] and ovine neutrophils [12,13]. In one
study with human neutrophils, suitable reference genes
included a TATA box binding protein (TBP), beta-actin
(ACTB), and succinate dehydrogenase complex subunit
A (SDHA) [11]. In another human study, guanine nu-
cleotide binding protein, ß-peptide 2-like1 (GNB2L1),
hypoxanthine phosphoribosyl-transferase 1 (HPRT), ri-
bosomal protein L32 (RPL32), ACTB, and beta-2-micro-
globulin (B2M) were suggested as suitable reference
genes in gene expression studies of neutrophils [10]. In
sheep neutrophils, we found that SDHA and glucose-6-
phosphate dehydrogenase (G6PD) in healthy sheep, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)

W. R. Vorachek et al. / Advances in Bioscience and Biotechnology 4 (2013) 6-14 7
and tyrosine 3-monoxygenase/tryptophan 5-monooxy-
genase activation protein zeta polypeptide (YWHAZ) in
foot rot diseased sheep were the best pairs of references
genes [13]. In ovine whole blood, SDHA and YWHAZ
were considered the most suitable reference genes as they
were stably expressed regardless of disease status [12].
In the bovine, potential reference genes have been evalu-
ated in polymorphonuclear leukocytes [14], mammary epi-
thelial cells [15], adipose tissue [16], milk somatic cells
[17], and WB-cells [18] of lactating dairy cows, and in
muscular tissue [19]; however, to our knowledge, poten-
tial reference genes have not been evaluated in WB-neu-
trophils under conditions of Se supplementation in grow-
ing beef calves.
Therefore, we investigated 11 potential neutrophil ref-
erence genes (Tabl e 1) including: ACTB, B2M, G6PD,
GAPDH, glycophorin C (GYPC), HPRT, phosphogly-
cerate kinase 1 (PGK1), ribosomal protein L19 (RPL19),
SDHA, transferrin receptor (TFRC), and YWHAZ. We
assessed gene expression levels, and analyzed gene ex-
pression stability using the programs geNorm [20], Norm
Finder [21], BestKeeper [22], and a comparative delta
Cq method [23], in WB neutrophils from beef calves,
males and females, with or without Se supplementation,
and in both treatment groups combined.
2. MATERIALS AND METHODS
2.1. Candidate Genes for Expression Studies
Eleven genes were selected from commonly used refer-
ence genes [11]: ACTB, B2M, G6PD, GAPDH, GYPC,
HPRT, PGK1, RPL19, SDHA, TFRC, and YWHAZ. The
length of the primers ranged from 18-bp to 27-bp, GC
content varied from 45% to 60%, and the expected PCR
product size was 71-bp to 126-bp (Table 2). Table 2 sum-
marizes primer information including R2 and efficiency
of qPCR analysis. The Minimum Information for Publi-
cation of Quantitative Real-Time PCR Experiments gui-
delines (MIQE) were followed for sample handling and
analysis [24] and a checklist is available in the supple-
mentary file.
Reference genes were selected based upon a literature
review to find genes that covered a wide variety of func-
tions and pathways: ACTB, GAPDH, and B2M [19];
HPRT, SDHA, and YWHAZ [14], RPL19 [25], and four
commonly used ovine neutrophil reference genes (G6PD,
TFRC, PGK1, and GYPC) [12,13]. All primers were
synthesized by Sigma Aldrich (St. Louis, MO) and puri-
fied by desalting. To verify that these primers were bo-
vine specific, each primer pair was used to amplify bo-
vine cDNA (AccuPrime Taq; Applied Biosystems, Foster
Table 1. Candidate reference genes evaluated in this study.
Gene symbol Gene name Function Accession number* Gene synonyms
ACTB Beta-actin Cytoskeletal structural protein NM_173979.3 Actin cytoplasmic 1;
beta-actin
B2M Beta-2-microglobin Beta-chain of class I major
histocompatibility complex molecules NM_173893.3
G6PD Glucose-6-phosphate
dehydrogenase Enzyme in carbohydrate metabolism NM_001244135.1
GAPDH Glyceraldehyde-3-phosphate
dehydrogenase Enzyme in carbohydrate metabolism NM_001034034.2 GAPD; G3PDH
GYPC Glycophorin C Integral RBC membrane binding protein NM_001002886.1 BOS_1916; CD236;
D236R
HPRT Hypoxanthine
phosphoribosyl-transferase 1
Phosphoribosyl-transferase (PRT)-type I
domain NM_001034035.2 HPRT1
PGK1 Phosphoglycerate kinase I
Catalyzes the transfer of the high-energy
phosphate group of 1,3-bisphosphoglycerate
to ADP, forming ATP and 3-phosphoglycerate
NM_001034299.1
RPL19 Ribosomal protein L19 Found in the large ribosomal subunit NM_001040516.1
SDHA Succinate dehydrogenase complex,
subunit A Mitochondrial respiratory chain NM_174178.2
TFRC Transferrin receptor Protease-associated domain containing
proteins, like transferrin receptors NM_001206577.1 p90; CD71
YWHAZ Tyrosine 3-monoxygenase/
tryptophan 5-monooxygenase
activation protein, zeta polypeptide
Signal transduction by binding to
phosphoserine containing proteins NM_174814.2
14-3-3 protein zeta/delta;
tyrosine
3-monooxygenase
*NCBI Reference Sequence database http://www.ncbi.nlm.nih.gov/RefSeq/.

W. R. Vorachek et al. / Advances in Bioscience and Biotechnology 4 (2013) 6-14 8
Table 2. Characteristics of the primers of the 11 candidate reference genes used for qPCR analysis.
Gene symbol Primer sequences (forward/reverse) Spanned ExonsAmplicon size (bp) R2 PCR Efficiency
ACTB AAGGCCAACCGTGAGAAGATGACC
TGTCACGCACAATTTCCCGCTC
2nd
3rd 97 0.98 0.94
B2M CTGTCGCTGTCTGGACTGG
TTTGGCTTTCCATCTTCTGG
1st
2nd 86 1.00 1.02
G6PD TGACCTATGGCAACCGATACAA
CCGCAAAAGACATCCAGGAT
10th
11th 76 1.00 1.03
GAPDH ATGCCTCCTGCACCACCA
AGTCCCTCCACGATGCCAA
7th
8th 86 0.99 1.35
GYPC ATCAACATCGCTGTCATTGC
CTCGTTGGTGTCCTATGTGC
3rd
4th 117 0.97 1.55
HPRT TTTATTCCTCATGGACTTAATTATGGA
CCACCCATCTCCTTCATCAC
4th
5th 71 0.99 1.02
RPL19 AGCCTGTGACTGTCCATTCC
ACGTTACCTTCTCGGGCATT
2nd
3rd 126 0.99 1.00
PGK1 ACTCCTTGCAGCCAGTTGCT
AGCACAAGCCTTCTCCACTTCT
3rd
4th 101 1.00 0.87
SDHA CATCCACTACATGACGGAGCA
ATCTTGCCATCTTCAGTTCTGCTA
4th
5th 90 0.98 1.74
TFRC TTCTGGGCAGACCTCAAATC
CAGCTTCACGTGGGACATAA
4th
5th 106 0.91 0.99
YWHAZ TGTAGGAGCCCGTAGGTCATCT
TTCTCTCTGTATTCTCGAGCCATCT
2nd
3rd 123 1.00 0.89
City, CA). The following conditions were employed:
94˚C for 2 min; then 35 cycles of 94˚C for 30 s, 60˚C for
30 s, and 68˚C for 30 s. The PCR products were then
purified using a QIAquick PCR Purification Kit (Qiagen
Sciences, Germantown, MD) according to manufactur-
er’s instructions, and quantitated using an ND-1000 Na-
noDrop Spectrophotometer (Thermo Fisher Scientific;
Waltham, MA). The PCR products were subsequently
sequenced at the Oregon State University Center for Ge-
nome Research and Biocomputing (Corvallis, OR) using
an automated ABI 3100 capillary sequencer (Applied Bi-
osystems). The sequences obtained were used in a
BLAST search (National Center for Biotechnology In-
formation, US National Library of Medicine, Bethesda,
MD) to verify that the products were bovine in origin
and were amplifying genes of interest.
2.2. Whole- Blood Collection
Calves were bled after feeding Se-enriched alfalfa hay
for 6 or 7 weeks, as part of a study that has been previ-
ously reported [26]. The experimental protocol was re-
viewed and approved by the Oregon State University
Animal Care and Use Committee. Jugular venous blood
was collected into evacuated ethylenediaminetetraacetic
acid (EDTA) tubes (10 mL; final EDTA concentration 2
g/L; Becton Dickinson, Franklin Lakes, NJ) and stored
on ice until transported to the lab for neutrophil isolation.
2.3. Neutrophil Isolation
Neutrophils were isolated within 4 h of collection, using
a Percoll (Sigma-Aldrich, St. Louis, MO) gradient tech-
nique, then resuspended in 1× Hank’s balanced saline so-
lution (HBSS; Life Technologies, Grand Island, NY) plus
0.5% fetal bovine serum (FBS; Life Technologies). Cells
were counted using a Coulter counter (Beckman Coulter,
Indianapolis, IN) to determine cell concentration. Briefly,
10 mL of anticoagulated blood was transferred into 50-
mL tubes (Thermo Fisher Scientific) and centrifuged at
1000 × g in a TJ-6 swinging bucket centrifuge (Beckman
Coulter) for 20 min at 22˚C. The plasma, buffy coat, and
one third of the RBC pack from each tube were asepti-
cally removed. The remaining RBC pack and leukocytes
were mixed with 34-mL ice-cold PBS (Life Technologies)
and layered onto 10 mL of freshly prepared 1.084 g/mL
Percoll. Tubes were centrifuged at 400 × g for 40 min at
22˚C. After centrifugation, RBC and neutrophils pelleted
at the bottom of the tube and the mononuclear cell band
remained at the sample/medium interface.
Neutrophils were isolated after removing the mononu-
clear cell band; all layers above the RBC pack were as-
pirated and discarded. The RBC were lysed using 24-mL
ice-cold hypotonic lysis buffer (10.56 mM Na2HPO4,
2.67 mM NaH2PO4, pH 7.3) for 90 s, and then isotonicity
was restored by adding 12-mL ice-cold hypertonic re-
store buffer (10.56 mM Na2HPO4, 2.67 mM NaH2PO4,
0.43 M NaCl, pH 7.3) to stop lysis. Neutrophils were
pelleted by centrifugation of tubes at 800 × g for 5 min at
22˚C in a TJ-6 centrifuge. The lysis solution was de-
canted, and the neutrophils were resuspended and wash-
ed twice more with 1× HBSS plus 0.5% FBS. The neu-

W. R. Vorachek et al. / Advances in Bioscience and Biotechnology 4 (2013) 6-14 9
trophils were then resuspended in 0.25 mL of 1× HBSS
with 0.5% FBS and stored on ice until needed. A 20-µL
aliquot of the cell suspension was used to determine cell
concentration using a Coulter counter (Beckman). An-
other 5-µL aliquot was used to assess purity of neutrophil
preparations (differential cell count) by microscopic exa-
mination after Wright-Giemsa staining (96% ± 1% neu-
trophils; mean ± SEM). The remaining neutrophils were
pelleted by centrifugation at 700 × g for 10 min at 4˚C in
a TJ-6 swinging bucket rotor (Beckman), supernatant
was removed, and cells were frozen at −80˚C.
2.4. RNA Isolation
Total RNA was extracted from previously frozen neu-
trophils using an RNeasy® Mini Kit (Qiagen Sciences)
according to manufacturer’s instructions. Genomic DNA
was eliminated with an RNase-Free DNase (Qiagen Sci-
ences) digestion. Total isolated RNA was quantified us-
ing an ND-1000 NanoDrop Spectrophotometer (Thermo
Fisher Scientific). Only samples with A260/A280 ratios be-
tween 1.40 and 2.16 were analyzed further. Samples were
stored at −80˚C until needed.
2.5. Quantitative PCR
Expression level of references genes in neutrophils was
determined by qPCR. First-strand cDNA was synthesized
from 50 ng of total RNA from each sample using the
High Capacity cDNA Reverse Transcription Kit (Applied
Biosystems). Subsequent qPCR was conducted using RT2
SYBR Green qPCR Mastermix (Qiagen Sciences) in 96-
well MicroAmp® reaction plates (Applied Biosystems)
with a final reaction volume of 25 μL. Each well con-
tained 12.5 μL of qPCR Mastermix, 1 μL of each primer,
1 μL of cDNA sample, and 9.5 μL of PCR-grade water.
Abundance of gene transcripts was determined by abso-
lute qPCR using the 7300 Real-time PCR System (Appli-
ed Biosystems). A negative control (containing all rea-
gents except target DNA) was included to verify the ab-
sence of contamination in each qPCR assay. Each reac-
tion consisted of the following steps: 10 min initial de-
naturation at 95˚C to activate the polymerase; followed
by 40 cycles of 15 s denaturation at 95˚C and 1 min an-
nealing-elongation at 60˚C. For each primer, PCR effi-
ciency and R2 were calculated using a standard curve de-
rived from a pooled cDNA mixture serially diluted 4-fold
over 4 measuring points, with two replications at each
measuring point.
2.6. Statistical Analysis of Neutrophil Gene
Expression Stability
The Cq values were reported as mean, standard deviation
(SD), and range. Expression stability of potential neu-
trophil reference genes was evaluated using generally
accepted Excel-based software tools [24] according to
instructions provided by the program developers, i.e.,
geNorm [20], NormFinder [21], BestKeeper [22], and a
comparative delta Cq method [23]. Ranking of potential
neutrophil reference genes was performed for neutrophils
collected from calves fed alfalfa hay that was not en-
riched with Se (calves consumed 0.95 mg Se/head/day)
and calves fed Se-enriched alfalfa hay (calves consumed
18.76 mg Se/head/day). To provide a summary statistic
for the four algorithms, the geometric mean of the rank-
ing for each of the four algorithms was calculated [27];
the gene with the lowest value was viewed as the most
stable reference gene. Normality of gene expression was
tested using the Shapiro Wilk statistic in SAS, version
9.2 (SAS, Inc., Cary, NC, USA) software. Group aver-
ages of Cq values were compared overall, between male
and female calves, between calves fed alfalfa hay that was
Se-enriched or not, and between blood samples taken on
two different dates (PROC GLM). The R2 and efficiency
were calculated as previously described [24]. All tests
were two-sided. Statistical significance was declared at P
≤ 0.05.
3. RESULTS AND DISCUSSION
3.1. Expression Level of Neutrophil Reference
Genes Evaluated in This Study
To identify the most appropriate set of reference genes for
gene expression analyses in WB neutrophils of growing
beef calves in the context of Se supplementation, a sys-
tematic investigation of 11 commonly used reference
genes (Table 1) was performed by qPCR experiments.
The 11 potential reference genes covered a variety of
pathways to avoid bias from co-regulation among them
[20]. The observed Cq values were distributed over a wide
range in beef calf neutrophils (Table 3), including highly
expressed B2M (Cq ± SD, 17.61 ± 0.62; Cq range, 2.62)
and less transcribed TFRC (29.83 ± 0.88; Cq range, 3.22)
and GYPC (31.52 ± 0.78; Cq range, 2.80). The least va-
riation was associated with YWHAZ (22.49 ± 0.35; Cq
range, 1.23) and the most variation was associated with
ACTB (22.44 ± 1.06; Cq range, 3.85). Three genes did not
pass the test for normal distribution at P ≤ 0.01, namely
SDHA (Shapiro-Wilk W = 0.87; P = 0.008), B2M (Sha-
piro-Wilk W = 0.82; P = 0.001), and HPRT (Shapiro-
Wilk W = 0.87; P = 0.007). None of the 11 genes evalu-
ated differed significantly by sex of calf or Se supplemen-
tation.
We arbitrarily selected a gene expression level >28
cycles or high variability (SD > 1.5 cycle) for exclusion of
potential reference genes from further consideration. This
eliminated GYPC and TFRC from further analysis. Our

W. R. Vorachek et al. / Advances in Bioscience and Biotechnology 4 (2013) 6-14 10
Table 3. Individual Cq values of the candidate reference genes
in weaned beef calves fed for 6 or 7 weeks 0.96 mg Se/head/day
(Control), 18.76 mg Se/head/day (High Selenium), or combined
groups.
Gene
symbol Calves, control (n = 10) Calves, high selenium (n = 12) Combined groups (n = 22)
Mean Cq SD Mean Cq SD Mean CqSD
ACTB 22.07 0.78 22.74 1.19 22.44 1.06
B2M 17.31 0.24 17.85 0.73 17.61 0.62
G6PD 21.21 0.59 21.74 0.75 21.50 0.72
GAPDH 19.96 0.73 20.18 0.74 20.08 0.73
GYPC 31.64 0.82 31.43 0.76 31.52 0.78
HPRT 27.74 0.49 27.20 0.83 27.44 0.73
PGK1 23.06 0.52 23.32 0.45 23.20 0.49
RPL19 20.85 0.48 20.90 0.52 20.88 0.49
SDHA 25.55 0.59 25.62 0.60 25.58 0.58
TFRC 30.05 0.78 29.64 0.95 29.83 0.88
YWHAZ 22.43 0.29 22.55 0.40 22.49 0.35
rationale was that the delta Cq for genes of interest com-
pared to reference genes in subsequent studies would be
more accurate if reference genes were expressed in suffi-
cient copy numbers to be reliably detected in all samples
and have limited variation.
3.2. GeNorm Analysis of Reference Genes
The program geNorm [20] provides a measure of gene
expression stability by calculating the average pairwise
variation of each reference gene from all the other refer-
ence gene candidates. In addition, it performs a ranking
of the candidate genes by stepwise exclusion of the worst
scoring gene and repeated recalculation of the average
gene expression stability value. The designers of geNorm
also stipulate that neither experimental conditions nor
cell type affects the expression ratio of a “true” reference
gene pair. This is based on the premise that the expres-
sion ratio of reference genes should be the same in all
experimental samples. Hence, expression ratios of gene
pairs were used as a measure of reference gene stability.
The stability values calculated by GeNorm were used to
rank gene expression in our study for potential reference
genes for bovine neutrophils (Table 4).
The lower the stability value, the more likely that a
candidate gene will be useful as a reference gene. Low
stability values indicate stable gene expression [20].
Genes with a stability level <0.5 are considered stable
[28]. Based on a stability level of <0.5 in geNorm analy-
sis, PGK1 (Cq range, 1.74), B2M (Cq range, 2.62),
GAPDH (Cq range, 2.73), YWHAZ (Cq range, 1.23),
and G6PD (Cq range, 3.18) would be suitable as refer-
ence genes in beef calves, independent of Se-supple-
Table 4. Stability ranking of candidate reference genes in
weaned beef calves fed for 6 or 7 weeks 0.96 mg Se/head/day
(Control), 18.76 mg Se/head/day (High Selenium), or combin-
ed groups, by the geNorm algorithm (lower stability values in-
dicate more stable gene expression).
Calves, control
(n = 10) Calves, high selenium (n = 12) Combined groups (n = 22)
Gene symbol and stability value
RPL19|SDHA 0.268 B2M|G6PD 0.397 PGK1|YWHAZ 0.355
G6PD 0.357 PGK1 0.419 B2M 0.444
PGK1 0.410 GAPDH 0.450 G6PD 0.467
YWHAZ 0.427 YWHAZ 0.481 GAPDH 0.483
B2M 0.445 RPL19 0.520 RPL19 0.517
GAPDH 0.491 SDHA 0.587 SDHA 0.558
HPRT 0.540 HPRT 0.709 HPRT 0.668
ACTB 0.656 ACTB 0.850 ACTB 0.794
mentation status. In contrast, four of the candidate refer-
ence genes would be excluded from consideration: ACTB
(Cq range, 3.85), HPRT (Cq range, 3.39), SDHA (Cq
range, 1.75), and RPL19 (Cq range, 1.83). Within the 5
acceptable genes B2M was not normally distributed. Si-
milar to our reference gene study for ovine neutrophils
[13], YWHAZ and GAPDH had suitable geNorm stabil-
ity values. Compared with GAPDH, YWHAZ had a smal-
ler Cq range, and better geNorm values (Table 4) for
control and combined groups.
Using multiple reference genes rather than a single
reference gene is likely to provide more robust and reli-
able results [20]. In the geNorm algorithm, the optimal
number of reference genes is determined when addition
of a further gene leads to a negligible reduction in the
average of gene stability estimates. For control calves,
the best pair of genes was RPL19|SDHA. For high Se
calves, the best pair of genes was B2M|G6PD; however,
B2M|G6PD was not normally distributed (Shapiro-Wilk
W = 0.87; P = 0.009). Overall the best pair of reference
genes was PGK1|YWHAZ (22.85 ± 0.39; Cq range,
1.36).
3.3. Norm Finder Analysis of Reference Genes
The Norm Finder program uses a model-based approach
that also estimates variation between sample subgroups.
The program analyzes inter- and intra-group expression
variation of potential reference genes. A stability value is
calculated based on analysis of gene expression data, and
the potential reference genes are then ranked. Lower va-
lues are assigned to the most stable genes. When Norm
Finder was used to identify potential neutrophil reference
genes in this study, YWHAZ, PGK1, and RPL19 ranked

W. R. Vorachek et al. / Advances in Bioscience and Biotechnology 4 (2013) 6-14 11
as the best choices (Table 5). The B2M gene did not rank
as high in the high Se calves or the combined groups.
Two of the candidate neutrophil reference genes would
be excluded from consideration as they ranked consis-
tently at or near the bottom four of the rank order: HPRT
and ACTB. The commonly used reference gene ACTB
was similarly a poor reference gene in WB cells of lac-
tating dairy cows [18]. The PGK1 |YWHAZ gene com-
bination had NormFinder stability values of 0.074 (con-
trol calves), 0.100 (high Se calves), and 0.089 (combined
groups).
3.4. BestKeeper Analysis of Reference Genes
The program BestKeeper estimates the expression stabil-
ity by performing a pairwise correlation analysis of each
pair of candidate gene Cq values. It then calculates the
geometric mean of the best suited genes. The weighted
index is correlated with up to ten target genes using the
same pairwise correlation analysis. When BestKeeper was
used to find potential reference genes (Table 6), YWHAZ,
PGK1, and RPL19 performed well in all groups, as did
the PGK1|YWHAZ combination (stability value 0.30 for
control calves and 0.32 for both high Se calves and com-
bined groups). B2M was near the bottom of the rank or-
der in Se-treated calves, in contrast to control calves. Si-
milar to geNorm and NormFinder analyses, ACTB would
be excluded from consideration as it ranked consistently
at or near the bottom of the rank order. The latter also
performed poorly as reference genes in WB cells of lac-
tating dairy cows [18].
3.5. Delta Cq Analysis of Reference Genes
Finally, a delta Cq analysis [23] was performed; this ana-
Table 5. Stability ranking of candidate reference genes in
weaned beef calves fed for 6 or 7 weeks 0.96 mg Se/head/day
(Control), 18.76 mg Se/head/day (High Selenium), or com-
bined groups, by the NormFinder algorithm (lower stability
values indicate more stable gene expression).
Calves, control
(n = 10) Calves, high selenium (n = 12) Combined groups (n = 22)
Gene symbol and stability value
YWHAZ 0.147 YWHAZ 0.128 YWHAZ 0.077
B2M 0.221 PGK1 0.199 PGK1 0.106
PGK1 0.228 RPL19 0.407 RPL19 0.389
RPL19 0.374 GAPDH 0.484 B2M 0.437
G6PD 0.393 B2M 0.513 GAPDH 0.505
SDHA 0.489 G6PD 0.558 G6PD 0.508
HPRT 0.563 SDHA 0.652 SDHA 0.572
GAPDH 0.565 HPRT 0.963 HPRT 0.899
ACTB 0.993 ACTB 1.241 ACTB 1.138
Table 6. Stability ranking of candidate reference genes in
weaned beef calves fed for 6 or 7 weeks 0.96 mg Se/head/day
(Control), 18.76 mg Se/head/day (High Selenium), or combin-
ed groups, by the BestKeeper algorithm (lower stability values
indicate more stable gene expression).
Calves, control
(n = 10) Calves, high selenium (n = 12) Combined groups (n = 22)
Gene symbol and stability value
B2M 0.19 YWHAZ 0.33 YWHAZ 0.28
YWHAZ 0.22 PGK1 0.39 PGK1 0.39
RPL19 0.36 RPL19 0.42 RPL19 0.40
PGK1 0.38 HPRT 0.52 B2M 0.46
HPRT 0.43 G6PD 0.54 HPRT 0.50
G6PD 0.49 GAPDH 0.54 SDHA 0.54
SDHA 0.50 SDHA 0.56 G6PD 0.55
ACTB 0.59 B2M 0.57 GAPDH 0.56
GAPDH 0.59 ACTB 0.94 ACTB 0.84
lysis is similar to the geNorm program in that pairs of
genes are compared using Cq differences. The compara-
tive delta Cq method compares the Cq value differences
between two reference genes from different samples, and
if the delta Cq value between pairs of genes remains con-
stant for all samples tested, then those reference genes
are either stably expressed or co-regulated. Potential re-
ference gene candidates were compared in this study and
ranked based on Cq value differences to determine those
with the least variance. Results are shown in Table 7.
The best choices for neutrophil reference genes were
PGK1, YWHAZ, and RPL19. In our reference gene study
for ovine neutrophils, YWHAZ and RPL19, along with
G6PD and GAPDH, had the lowest delta Cq stability
values [13]. Similar to geNorm, Norm-Finder, and Best-
Keeper analyses, ACTB would be excluded as a refer-
ence gene from consideration as it ranked consistently at
or near the bottom four of the rank order. The PGK1|
YWHAZ combination performed best with stability val-
ues of 0.45 (control calves), 0.55 (high Se calves), and
0.52 (combined groups).
3.6. Stability of Neutrophil Reference Genes
Evaluated in This Study
Currently, there is no consensus as to what is the best
program to evaluate potential reference genes. Therefore,
we calculated the geometric mean of expression stability
by four statistical approaches using the Geomean pro-
gram. We found good agreement between the results with
the four statistical approaches. The neutrophil reference
genes, YWHAZ, PGK1, and RPL19, were consistently
ranked among the top three most stable genes by all four

W. R. Vorachek et al. / Advances in Bioscience and Biotechnology 4 (2013) 6-14 12
programs (Table 8). Several conventional reference genes
proved to be less reliable in neutrophils from beef calves,
specifically ACTB, GYPC, HPRT, and TFRC.
Vandesompele et al. [20] suggested that normalization
should be based on multiple references genes rather than
a single gene to provide more robust and reliable results.
The PGK1|YWHAZ combination performed best with
Geomean values of 1.57 (control calves) and 1.00 (high
Se calves) and 1.19 (combined groups), which is similar
Table 7. Stability ranking of candidate reference genes in
weaned beef calves fed for 6 or 7 weeks 0.96 mg Se/head/day
(Control), 18.76 mg Se/head/day (High Selenium), or combin-
ed groups, by the comparative delta Cq method (lower stability
values indicate more stable gene expression).
Calves, control
(n = 10) Calves, high selenium (n = 12) Combined groups (n = 22)
Gene symbol and stability value
YWHAZ 0.51 PGK1 0.62 PGK1 0.60
PGK1 0.54 YWHAZ 0.67 YWHAZ 0.62
B2M 0.54 RPL19 0.72 RPL19 0.68
G6PD 0.59 GAPDH 0.77 B2M 0.71
RPL19 0.59 B2M 0.77 G6PD 0.73
SDHA 0.64 G6PD 0.78 GAPDH 0.75
GAPDH 0.71 SDHA 0.86 SDHA 0.78
HPRT 0.73 HPRT 1.11 HPRT 1.04
ACTB 1.06 ACTB 1.34 ACTB 1.24
Table 8. Stability ranking of candidate reference genes in
weaned beef calves fed for 6 or 7 weeks 0.96 mg Se/head/day
(Control), 18.76 mg Se/head/day (High Selenium), or com-
bined groups, by the Geomean of ranking values for geNorm,
NormFinder, BestKeeper and the comparative delta Cq method
(lower stability values indicate more stable gene expression).
Calves, control
(n = 10) Calves, high selenium (n = 12) Combined groups (n = 22)
Gene symbol and stability value
YWHAZ 1.78 YWHAZ 1.78 YWHAZ 1.19
B2M 2.45 PGK1 1.86 PGK1 1.41
RPL19 2.78 RPL19 3.57 RPL19 3.57
PGK1 3.13 G6PD 3.66 B2M 3.72
SDHA 3.98 B2M 3.76 G6PD 5.38
G6PD 4.36 GAP DH 4.43 GAPDH 5.89
HPRT 6.88 HPRT 6.73 SDHA 6.74
GAPDH 7.71 SDHA 7.00 HPRT 7.11
ACTB 8.74 ACTB 9.00 ACTB 9.00
or better than any single gene (Table 8). Whereas PGK1
is an enzyme that is involved in glycolysis, YWHAZ
plays a role in signal transduction by binding to phos-
phoserine containing proteins and is involved in many
vital cellular processes, including metabolism, protein traf-
ficking, apoptosis and cell cycle regulation [29]. More
reliable qPCR data normalization should be achieved us-
ing a combination of genes from different biological path-
ways, rather than a single gene.
Our selection of reference genes for bovine neutro-
phils differs from results obtained for human neutrophils
whereby recommended reference genes included TBP,
ACTB, and SDHA [11] or GNB2L1, HPRT1, RPL32,
ACTB, and B2M [10], and also differs from results we
obtained for sheep neutrophils, whereby SDHA|G6PD
and GAPDH|YWHAZ were the best pairs of references
genes [13]. In lactating dairy cows, YWHAZ along with
SDHA and 18S rRNA, were considered the most suitable
reference genes for polymorphonuclear leukocytes [14].
In another lactating dairy cow study that investigated
milk somatic cells, which consist predominantly of leu-
kocytes, including lymphocytes, neutrophils (5% to 25%)
and macrophages, the most stable reference genes in-
cluded ACTB and GAPDH [17]. These results under-
score the need to validate neutrophil reference genes un-
der different experimental conditions.
4. CONCLUSION
A literature search for candidate genes in the species and
cell type of interest provides a starting point for reference
gene selection. Once potential reference genes have been
screened for expression level and overall variability, can-
didate genes can be further analyzed for expression sta-
bility using four readily available software algorithms:
geNorm, NormFinder, BestKeeper, and the comparative
delta Cq method. The WB-neutrophil reference genes
YWHAZ, PGK1, and RPL19 consistently ranked among
the top four most stable genes under our experimental
conditions. Several conventional reference genes proved
to be less reliable, including ACTB, GYPC, HPRT, and
TFRC. Multiple references genes rather than a single gene
may provide more robust and reliable results. The best
pair of reference genes in whole-blood neutrophils from
beef calves was PGK1|YWHAZ.
5. ACKNOWLEDGEMENTS
Funded in part by Animal Health and Disease Project Formula Funds
and the Agricultural Research Foundation, Oregon State University,
Corvallis, OR 97331-4802, USA (J.A. Hall, Principal Investigator).
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