GENE-40166; No. of pages: 7; 4C: Gene xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
3Q3
Sang-Je Park a,⁎,1, Seul Gi Kwon b,1, Jung Hye Hwang b, Da Hye Park b, Tae Wan Kim b, Chul Wook Kim b,⁎⁎
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Article history: Received 19 August 2014 Received in revised form 22 December 2014 Accepted 25 December 2014 Available online xxxx
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Keywords: Berkshire Duroc Landrace Normalization Reference gene RT-qPCR Yorkshire
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National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk 363-883, Republic of Korea Swine Science and Technology Center, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea
a b s t r a c t
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Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most reliable molecular biology technique for assessment of mRNA expression levels. However, to obtain the accurate RT-qPCR results, the expression levels of genes of interest should be normalized with appropriate reference genes and optimal numbers of reference genes. In this study, we assessed the expression stability of 15 well-known candidate reference genes (ACTB, ALDOA, B2M, GAPDH, HPAR1, HSPCB, PGK1, POLR2G, PPIA, RPL4, RPS18, SDHA, TBP, TOP2B, and YWHAZ) in seven body tissues (liver, lung, kidney, spleen, stomach, small intestine, and large intestine) of Berkshire, Landrace, Duroc, and Yorkshire pigs using three excel-based programs, geNorm, NormFinder, and BestKeeper. Combination analysis of these three programs showed that the stable and appropriate reference genes are PPIA, TBP, and HSPCB in Berkshire pigs; PPIA, TBP, RPL4, and RPS18 in Landrace pigs; PPIA and TBP in Duroc pigs; and PPIA, TOP2B, RPL4, and RPS18 in Yorkshire pigs. Because the four pig breeds had different suitable reference genes, the selection of appropriate reference genes is essential in RT-qPCR analyses. Taken together, our data could help to select reliable reference genes for the normalization of expression levels of various target genes in pigs. © 2014 Published by Elsevier B.V.
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1. Introduction
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Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is one of the most frequently used experimental methods for the evaluation of mRNA expression levels due to its sensitivity, specificity, accuracy, and cost-effectiveness. However, quantification of mRNA levels using RT-qPCR may be affected by various parameters, including RNA quality and purity in RNA extraction steps, differing sample amounts, and enzymatic efficiency in reverse transcription steps, and PCR efficiency (Vandesompele et al., 2002; Park et al., 2013). Therefore, normalization processes should be performed
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Abbreviations: AF, African green monkeys; AS, alternative splicing; BCS1L, BC1 (ubiquinol-cytochrome c reductase) synthesis-like (BCS1L); BO, bonobos; CA, capuchins; CDS, coding sequence; CH, chimpanzees; CO, colobus monkeys; CR, crab-eating monkeys; HERV, human endogenous retrovirus; HU, humans; JA, Japanese monkeys; LA, langurs; LINE, long interspersed element; MA, mandrills; MAR, marmosets; NI, night monkeys; NWM, New World monkey; OWM, Old World monkey; PI, pig-tail monkeys; PPT, polypyrimidine tract; RH, rhesus monkeys; RL, ring-tailed lemurs; SINE, short interspersed element; SP, spider monkeys; SQ, squirrel monkeys; TA, tamarins; TEs, transposable elements; TSS, transcriptional start site; UTR, untranslated region ⁎ Corresponding author. ⁎⁎ Correspondence to: C. W. Kim, Department of Animal Resources Technology, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea. E-mail addresses: [email protected] (S.-J. Park), [email protected] (C.W. Kim). 1 These authors contributed equally to this work.
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Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs
1Q2
using constitutively expressed genes, i.e., reference genes or internal control genes. In previous studies, housekeeping genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), and ribosomal protein s18 (RPS18), were used to normalize mRNA expression without selection of appropriate reference genes. However, these genes have variable expression levels across tissue types, disease state, and different experimental conditions (Vandesompele et al., 2002; Yperman et al., 2004; Maroufi et al., 2010; Beekman et al., 2011). Therefore, to obtain an accurate quantification of target genes, selection of suitable and stable reference genes among the various candidate reference genes should be performed according to breed, tissue and cell type, and experimental conditions. The pig (Sus scrofa) is an economically important livestock animal for meat production. It is also an ideal animal model for the study of human disease due to its similar genome size and organization (Hasler-Rapacz et al., 1995; Uddin et al., 2011). Therefore, numerous quantitative analyses of target genes have been performed using RT-qPCR experiments in various pig breeds to improve meat quality and to study human diseases (Henriksen et al., 2009; Li et al., 2012; Xiao et al., 2012; Bruun et al., 2013). To our knowledge, several reference gene studies have been performed in various pig tissue samples, including fat-type tissues, muscle-type tissues, articular cartilage, backfat tissues, longissimus dorsi muscle, peripheral blood mononuclear cells (PBMC), different embryonic stage samples, epithelial endometria, cardiac tissue, and others in various purebred and hybrid pig breeds
http://dx.doi.org/10.1016/j.gene.2014.12.052 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
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2.1. Ethics statement
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The Animal Care and Use Committee of GNTECH (Gyeongnam National University of Science and Technology) specifically waived the need for consent because approval by the ethics committee is not required for the slaughter of farm animals in the Republic of Korea. Used farm pigs were generated from purebred breeding pigs and these pigs were tested and regulated at the First Korea Swine Testing Station. All pigs were slaughtered in a commercial abattoir (Bukyung livestock joint market). Slaughterhouse management gave the necessary permissions for the tissue collection. Pigs used in this study were slaughtered in accordance with the guidelines on animal care and use established by the Republic of Korea's Animal Protection Act and related laws. Four Pig breeds weighing approximately 110 kg (average of age 180 days) were transported to an abattoir near the experimental station. They were slaughtered by stunning with electrical tongs (300 V for 3 s) after 12 h of feed restriction. The shocked pigs were exsanguinated while being hanged.
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2.2. Total RNA isolation and cDNA preparation
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After exsanguination, seven different body tissues (liver, lung, kidney, spleen, stomach, small intestine, and large intestine) of Berkshire (n = 3, male), Landrace (n = 3, male), Duroc (n = 3, male), and Yorkshire (n = 3, male) pigs were directly collected, and all samples were immediately snap-frozen and stored in liquid nitrogen, prior to total RNA extraction. In this study, we investigated selection of reference genes using three male pigs in each breeds. Because, almost slaughtered purebred pigs were male pigs, and female purebred pigs were used for breeding in Korea farm. Therefore, we first analyzed using male pig breeds. Total RNA was extracted from seven different body tissues using the RNeasy Plus Mini kit (Qiagen) following the manufacturer's instructions. In this kit, a gDNA Eliminator spin column was used to remove DNA contamination from the total RNA preparations. Total RNA was quantified using a NanoDrop® ND-1000 UV–vis Spectrophotometer. Reverse transcription was performed using SuperScript™ II Reverse Transcriptase (Invitrogen) with RNase OUT (Invitrogen) following the manufacturer's instructions. To remove RNA complementary to the cDNA, RNase H (Invitrogen) was used. We performed PCR amplification without the reverse transcription (RT) reaction using pure RNA
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Specific primer pairs for candidate reference genes were designed using the Primer3 program (http://frodo.wi.mit.edu/primer3/; Table 1) (Rozen and Skaletsky, 2000). BLAST searches were performed to confirm gene specificity of the primer sequences, and the results showed the absence of multi-locus matching at individual primer sites. Most primers spanned at least 2 exons or had a large intron sequence between the forward and reverse primers to avoid falsepositive amplification of contaminating genomic DNA in the RNA samples. The nucleotide sequences of the RT-PCR products for the 15 candidate reference genes were obtained using standard molecular cloning and sequencing procedures (Fig. S1). Amplification efficiencies and correlation coefficients (R2 values) of the 15 genes were generated using the slopes of the standard curves obtained from serial dilutions. Standard curves from a 10-fold dilution series were used to calculate the amplification efficiency (Table 1). The amplification efficiency was calculated according to the formula: efficiency (%) = (10(−1/slope) − 1) × 100. The efficiency range of the real-time RT-PCR amplifications for all the tested genes was 86% to 106%.
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2.4. Real-time RT-PCR amplification
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Real-time RT-PCR using SYBR Green was performed using a Rotor Gene-Q thermocycler (Qiagen). In each run, 1 μL of cDNA was used as the template for each reaction. The samples were added to 19 μL of the reaction mixture (7 μL H2O, 10 μL Rotor Gene SYBR Green PCR mastermix (Qiagen), and 1 μL each of the forward and reverse primers). Real-time RT-PCR amplification of the 15 genes was performed in 40 cycles of 94 °C for 5 s and 60 °C for 10 s. The amplification specificity of each RT-qPCR assay was confirmed by melting curve analysis. The temperature range for the analysis of the melting curves was 70 °C–95 °C for 5 s. As shown in Fig. S2, each primer pair showed a single, sharp peak, indicating that the primers amplified a single, specific PCR product. Amplification was not detected in the no-template controls (NTC) for all candidate reference genes (Table 1). These experiments were performed at least three times.
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2.5. Characterization of the analysis programs
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The geNorm program (Vandesompele et al., 2002) provides a measure of gene expression stability (M value), which is the mean pair-wise variation between an individual gene and all other tested control genes. This method differs from model-based approaches because it compares genes based on the similarity of their expression profiles. Cq values are converted to scale expression quantities using the ΔCq method and are recorded in the geNorm program, which then ranks the genes based on their M values; the genes with high M values are less stably expressed and would make bad reference genes and those with low M values are stably expressed and would make good reference genes. In addition, the geNorm program can calculate the optimal number of required reference genes for obtaining reliable results from RT-qPCR studies. This calculation was performed by analysis of the pair-wise variation (V value) of sequential normalization factors (NF) with an increasing number of reference genes (NFn and NFn + 1). NormFinder (Andersen et al., 2004) is a tool to identify optimally stable reference genes through the determination of expression stabilities using a model-based approach in Microsoft Excel. This program focuses on finding the two genes with the least intra- and inter-group expression variation or the most stable reference gene in intra-group expression variation. In this program, genes with lower stability values
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samples (no-RT control) and determined that the prepared mRNA 133 samples did not contain genomic DNA. 134
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(Erkens et al., 2006; Kuijk et al., 2007; Nygard et al., 2007; McBryan et al., 2010; Gu et al., 2011; Li et al., 2011; Martino et al., 2011; Piorkowska et al., 2011; Uddin et al., 2011; Wang et al., 2011; Xiang-Hong et al., 2011; McCulloch et al., 2012). However, studies on the selection of appropriate reference genes have not yet been performed on body tissues of Berkshire, Landrace, Duroc, and Yorkshire pigs. Therefore, we selected suitable reference genes and performed a comparative analysis in these four pig breeds. The aim of this study was to select and evaluate the stability of fifteen candidate reference genes using seven body tissues of Berkshire, Landrace, Duroc, and Yorkshire pigs. The stabilities of ACTB, aldolase A, fructose-bisphosphate (ALDOA), β-2-microglobulin (B2M), GAPDH, hypoxanthine phosphoribosyltransferase 1 (HPRT1), heat shock 90 kDa protein 1, beta (HSPCB), phosphoglycerate kinase 1 (PGK1), peptidylprolyl isomerase A (cyclophilin A) (PPIA), polymerase (RNA) II (DNA directed) polypeptide G (POLR2G), ribosomal protein L4 (RPL4), RPS18, succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB), topoisomerase II beta (TOP2B), and tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein ζ polypeptide (YWHAZ) were analyzed using the geNorm (Vandesompele et al., 2002), NormFinder (Andersen et al., 2004), and BestKeeper (Pfaffl et al., 2004) programs.
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Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
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Table 1 Primers for the 15 candidate reference genes and parameters derived from RT-qPCR data analysis. Gene name
ACTB
Beta actin
ALDOA
Aldolase A, fructose-bisphosphate
B2M
Beta-2-microglobulin
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
NM_001206359.1
HPRT1
Hypoxanthine phosphoribosyltransferase 1
Martino et al. (2011)
HSPCB
Heat shock 90 kDa protein 1, beta
Gu et al. (2011)
PGK1
Phosphoglycerate kinase 1
Wang et al. (2011)
POLR2G
Polymerase (RNA) II (DNA directed) polypeptide G
XM_005660782.1
PPIA
Peptidylprolyl isomerase A (cyclophilin A)
Uddin et al. (2011)
RPL4
Ribosomal protein L4
Uddin et al. (2011)
RPS18
RIBOSOMAL protein S18
NM_213940.1
SDHA
Succinate dehydrogenase complex, subunit A
Erkens et al. (2006)
TBP
TATA box binding protein
Martino et al. (2011)
TOP2B
Topoisomerase II beta
NM_001258386.1
YWHAZ
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide
Erkens et al. (2006)
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Abbreviation
GenBank accession no. or reference
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N.d.: Not detected. a If a primer is located on 2 exons, the junctions are shown with a virgule. b No template control.
Primera
Exon(s)
Amplicon size (bp)
PCR efficiency (%)
R2
NTCb (Cq)
3rd 3rd/4th Unknown
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106
0.99761
N.d.
142
99
0.99933
N.d.
1st/2nd 3rd 4th 5th/6th 1st 2nd 8th 9th 2nd/3rd 3rd 4th/5th 7th 4th 5th 5th 6th/7th 2nd/3rd 4th/5th 5th 6th Unknown
166
105
0.99209
N.d.
130
102
0.99271
N.d.
181
93
0.99562
N.d.
131
98
0.9931
N.d.
126
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0.99957
N.d.
181
99
0.99831
N.d.
171
98
0.9982
N.d.
185
99
0.9833
N.d.
74
96
0.99792
N.d.
193
86
0.99861
N.d.
124
88
0.9975
N.d.
16th/17th 18th 4th 5th
115
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0.99526
N.d.
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0.99555
N.d.
Forward (F)/reverse (R) Martino et al. (2011)
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Gu et al. (2011)
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Martino et al. (2011)
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F: TCTGGCACCACACCTTCT R: TGAT/CTGGGTCATCTTCTCAC F: GAACCAACGGCGAGACAA R: ATGATGGCGAGGGAGGAG F: TTCA/CACCGCTCCAGTAG R: CCAGATACATAGCAGTTCAGG F: ATCCTGGGCTACACTGAGGA R: TGTCGTAC/CAGGAAATGAGCT F: CCGAGGATTTGGAAAAGGT R: CTATTTCTGTTCAGTGCTTTGATGT F: GGCAGAAGACAAGGAGAAC R: CAGACTGGGAGGTATGGTAG F: AGATAACGAACAACCAGAG/G R: TGTCAGGCATAGGGATACC F: CTCAAGTCAACAAG/GTCGGAC R: GTCCCAACAATCTTCAGGCG F: CACAAACGGTTCCCAGTTTT R: TGTCCACAGTCAGCAATGGT F: AGGAGGCTGTTCTGCTTCTG R: TC/CAGGGATGTTTCTGAAGG F: GCGATTAAG/GGTGTAGGACG R: GAC/CTGGCTGTACTTCCCAT F: GAACCGAAGATGGCAAGA R: CAGGAGATCCAAGGCAAA F: GATGGACGTTCGGTTTAGG R: AGCAGCACAGTACGAGCAA F: AA/GGGCGAGAGGTCAATGAT R: ACATCTTCTCGTTCTTGCGC F: ATGCAACCAACACATCCTATC R: GCATTATTAGCGTGCTGTCTT
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Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
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3. Results
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3.1. Selection of candidate reference genes
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To select suitable reference genes for the seven body tissues of four pig breeds, we chose 15 candidate reference genes based on previous studies in which commonly used reference genes were determined to be stably expressed. We also chose genes belonging to different functional classes to minimize the risk of coregulation. On the basis of these criteria, our candidate genes were ACTB, ALDOA, B2M, GAPDH, HPRT1, HSPCB, PGK1, PPIA, POLR2G, RPL4, RPS18, SDHA, TBP, TOP2B, and YWHAZ (Table 1) (Erkens et al., 2006; Kuijk et al., 2007; Nygard et al.,
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a) GeNorm analysis The stability values (M value) of the 15 candidate reference genes were calculated using the geNorm program (Fig. 1). PPIA and TBP were identified as the two most stable genes in Berkshire and Duroc pigs, with low M-values of 0.21 and 0.15, respectively. RPL4 and RPS18 had low M-values of 0.16 and 0.18 in Landrace and Yorkshire pigs, respectively, and were identified as the two most stable genes. In Berkshire pigs, the most stable genes following PPIA were HSPCB (0.23), RPS18 (0.24), RPL4 (0.27), POLR2G (0.36), B2M (0.43), PGK1 (0.50), TOP2B (0.56), GAPDH (0.61), YWHAZ (0.68), ALDOA (0.73), ACTB (0.76), SDHA (0.84), and HPRT1 (0.97). In Duroc pigs, the most stable genes following TBP were RPL4 (0.25), HSPCB (0.36), RPS18 (0.41), TOP2B (0.46), B2M (0.50), ALDOA (0.53), POLR2G (0.58), GAPDH (0.61), ACTB (0.66), YWHAZ (0.69), PGK1 (0.72), SDHA (0.80), and HPRT1 (0.90). In Landrace pigs, the most stable genes following RPL4 were PPIA (0.28), TOP2B (0.35), TBP (0.38), HSPCB (0.41), ALDOA (0.46), ACTB (0.50), YWHAZ (0.53), GAPDH (0.57), B2M (0.61), POLR2G (0.65), PGK1 (0.69), SDHA (0.76), and HPRT1 (0.87). In Yorkshire pigs, the most stable genes following RPS18 were PPIA (0.30), TOP2B (0.33), HSPCB (0.38), POLR2G (0.42), TBP (0.45), B2M (0.48), ACTB (0.52), ALDOA (0.56), YWHAZ (0.59), GAPDH (0.63), PGK1 (0.66), SDHA (0.75), and HPRT1 (0.87). According to Vandesompele et al. (the developers of the geNorm program), the use of a minimal number of the most stable reference genes is recommended for the calculation of the normalization
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All experiments were performed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., 2009).
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3.2. Expression stability of candidate reference genes
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2007; Gu et al., 2011; Li et al., 2011; Martino et al., 2011; Uddin et al., 218 2011; Wang et al., 2011; McCulloch et al., 2012). 219
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show less varied expressions and have a stably expressed pattern, and genes with higher stability values show more varied expressions and have the least stably expressed pattern. BestKeeper (Pfaffl et al., 2004) index is created using the geometric mean of each candidate gene's Cq values. This program determines the most stably expressed genes based on correlation coefficient (r) analysis for all pairs of candidate reference genes (≤10 genes) and calculates the percentage coefficient of variation (CV) and standard deviation (SD) using each candidate gene's crossing point (CP) value (the quantification cycle value; Cq). In this program, genes with higher r values and lower CV and SD values are stable reference genes.
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Fig. 1. Ranking of 15 candidate reference genes using the geNorm program. The average expression stability (M) of 15 candidate reference genes and the best combination of two genes were calculated for Berkshire pigs (A), Duroc pigs (B), Landrace pigs (C), and Yorkshire pigs (D). Lower M values indicate more stable expression.
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
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factor (NF) (Vandesompele et al., 2002). Therefore, we analyzed the optimal number of required reference genes for obtaining reliable results from RT-qPCR studies (Fig. 2). The original paper using the geNorm program proposed 0.15 as the cut-off value, which suggests that additional reference genes would be unnecessary if the V value was b0.15. The pair-wise variation V2/3 was 0.070 in Berkshire pigs, 0.098 in Duroc pigs, 0.110 in Landrace pigs, and 0.116 in Yorkshire pigs. All values are lower than 0.15; however, the V value decreased significantly with the addition of reference genes, except for Duroc pigs. Therefore, to calculate the NF more accurately, the two most stable reference genes were recommended for Duroc pigs, the
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Fig. 2. Determination of the optimal number of reference genes for normalization. The geNorm program calculated the normalization factor (NF) from at least two genes and the variable V defines the pair-wise variation between two sequential NF values. Berkshire pigs (A), Duroc pigs (B), Landrace pigs (C), and Yorkshire pigs (D).
Table 2 Gene stability value calculations by NormFinder.
t2:3
Berkshire
t2:4 t2:5
Gene name
Stability value
Gene name
Stability value
Gene name
Stability value
Gene name
Stability value
t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20
PPIA TBP HSPCB RPS18 RPL4 TOP2B GAPDH POLR2G PGK1 B2M ALDOA YWHAZ ACTB SDHA HPRT1
0.062 0.073 0.073 0.106 0.161 0.393 0.395 0.41 0.447 0.467 0.618 0.643 0.67 0.811 1.212
PPIA TBP RPL4 HSPCB TOP2B GAPDH POLR2G RPS18 B2M ALDOA PGK1 ACTB YWHAZ SDHA HPRT1
0.078 0.079 0.131 0.297 0.305 0.341 0.377 0.381 0.403 0.424 0.454 0.574 0.615 0.822 1.002
PPIA TBP RPL4 RPS18 TOP2B GAPDH HSPCB POLR2G ALDOA B2M PGK1 ACTB YWHAZ SDHA HPRT1
0.111 0.134 0.156 0.174 0.25 0.288 0.369 0.393 0.404 0.476 0.494 0.501 0.557 0.718 1.054
PPIA TOP2B POLR2G RPS18 RPL4 TBP HSPCB GAPDH B2M PGK1 ACTB ALDOA YWHAZ SDHA HPRT1
0.046 0.176 0.201 0.204 0.218 0.25 0.289 0.356 0.387 0.411 0.475 0.495 0.573 0.824 1.11
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Landrace
Yorkshire
three most stable reference genes were recommended for Berkshire and Yorkshire pigs, and the four most stable reference genes were recommended for Landrace pigs as the optimal numbers of reference genes. b) NormFinder analysis NormFinder calculates the stability value and standard error according to the expression variation of the candidate reference genes. Analysis of our NormFinder data showed that PPIA was the most stable reference gene with the lowest stability value in all pig breeds (Table 2). TBP (0.073), HSPCB (0.073), RPS18 (0.106), RPL4 (0.161), TOP2B (0.393), GAPDH (0.395), POLR2G (0.410), PGK1 (0.447), B2M (0.467), ALDOA (0.618), YWHAZ (0.643), ACTB (0.670), SDHA (0.811), and HPRT1 (1.212) had increasing stability values in Berkshire pigs; TBP (0.079), RPL4 (0.131), HSPCB (0.297), TOP2B (0.305), GAPDH (0.341), POLR2G (0.377), RPS18 (0.381), B2M (0.403), ALDOA (0.424), PGK1 (0.454), ACTB (0.574), YWHAZ (0.615), SDHA (0.822), and HPRT1 (1.002) had increasing stability values in Duroc pigs; TBP (0.134), RPL4 (0.156), RPS18 (0.174), TOP2B (0.250), GAPDH (0.288), HSPCB (0.369), POLR2G (0.393), ALDOA (0.404), B2M (0.476), PGK1 (0.494), ACTB (0.501), YWHAZ (0.557), SDHA (0.718), and HPRT1 (1.054) had increasing stability values in Landrace pigs; and TOP2B (0.176), POLR2G (0.201), RPS18 (0.204), RPL4 (0.218), TBP (0.250), HSPCB (0.289), GAPDH (0.356), B2M (0.387), PGK1 (0.411), ACTB (0.475), ALDOA (0.495), YWHAZ (0.573), SDHA (0.824), and HPRT1 (1.110) had increasing stability values in Yorkshire pigs. c) BestKeeper analysis The BestKeeper program is also an Excel-based software tool. On the basis of the correlation coefficients (r), CV, and SD values, the optimal reference gene was determined (Table 3). This program can calculate r values for up to 10 genes. Therefore, we selected
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
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Berkshire
t3:4
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PGK1
POLR2G
PPIA
RPL4
RPS18
TBP
TOP2B
7 16.05 0.55 3.45 0.966 0.001
7 17.58 0.74 4.22 0.837 0.019
7 18.30 0.39 2.13 0.616 0.141
7 14.36 0.50 3.50 0.993 0.001
7 14.26 0.45 3.18 0.946 0.001
7 13.61 0.69 5.06 0.986 0.001
7 20.90 0.53 2.52 0.966 0.001
7 19.54 0.79 4.02 0.854 0.014
ALDOA 16.24 0.83 5.13 0.851 0.015
B2M 13.12 0.76 5.80 0.861 0.013
GAPDH 15.62 0.73 4.67 0.805 0.029
HSPCB 16.03 0.66 4.13 0.911 0.004
POLR2G 18.20 0.49 2.71 0.679 0.093
PPIA 15.15 0.56 3.66 0.955 0.001
RPL4 14.26 0.45 3.17 0.992 0.001
RPS18 13.65 0.39 2.82 0.804 0.029
TBP 20.91 0.65 3.11 0.948 0.001
TOP2B 19.61 0.94 4.77 0.945 0.001
ACTB 15.15 0.73 4.79 0.974 0.001
ALDOA 16.34 0.58 3.52 0.948 0.001
GAPDH 15.31 0.38 2.51 0.349 0.444
HSPCB 15.75 0.43 2.72 0.815 0.026
PPIA 14.63 0.40 2.74 0.842 0.017
RPL4 14.15 0.27 1.90 0.914 0.004
RPS18 13.69 0.30 2.18 0.933 0.002
TBP 21.21 0.52 2.46 0.815 0.026
TOP2B 19.58 0.52 2.63 0.853 0.015
YWHAZ 17.30 0.70 4.06 0.930 0.002
ACTB 15.15 0.73 4.79 0.981 0.001
ALDOA 16.13 0.57 3.54 0.832 0.020
B2M 13.91 0.66 4.75 0.820 0.024
HSPCB 15.80 0.45 2.86 0.923 0.003
POLR2G 18.60 0.24 1.29 0.396 0.381
PPIA 14.64 0.31 2.11 0.840 0.018
RPS18 13.78 0.25 1.79 0.798 0.032
TBP 21.11 0.58 2.76 0.873 0.010
TOP2B 19.20 0.44 2.31 0.958 0.001
geo Mean std dev CV r p-Value Yorkshire
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n number of samples, geo Mean the geometric mean of Cq, std dev standard deviation of Cq, CV coefficient of variation, and r coefficient of correlation. High ranked genes were described in bold.
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the 10 best candidate genes using the results of the geNorm and NormFinder programs. In the BestKeeper program, genes with higher r value (≥0.900) and lower CV and SD values are stable and suitable reference genes. In Berkshire pigs, the POLR2G gene had the lowest CV (2.13) and SD (0.39) values among the 10 candidate reference genes, and was stably expressed across all tested samples. However, PORL2G had a low r value (0.616) with the other candidate reference genes, which suggests that its expression was not correlated with the expression patterns of the other candidate reference genes. Therefore, we selected TBP as the reference gene for Berkshire pigs because it was stably expressed with high r value (0.966) and low CV (2.52) and SD (0.53) values. TBP, RPL4, and TOP2B were selected as stable reference genes for Duroc, Landrace, and Yorkshire pigs, respectively.
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4. Discussion
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Gene expression analysis is the one of the most commonly and widely used strategies in molecular biology studies, and RT-qPCR has become the most effective method for quantifying gene expression due to its accuracy, specificity, and cost-effectiveness. However, RT-qPCR data can be influenced by normalization with different reference genes. Therefore, the selection of appropriate reference genes is essential for the reliable and reproducible assessment of gene expression. Indeed, many studies on the selection of suitable reference genes have been performed in various species, in different cell or tissue types, and under different experimental conditions. Currently, to understand the gene function and the associations between various genes, meat quality, and reproduction, quantitative analyses have been performed using RT-qPCR in different body tissues and embryos of pigs (Davoli et al., 2011; Huang et al., 2012; Larsen et al., 2012; Li
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RPL4 14.41 0.23 1.62 0.845 0.017
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geo Mean std dev CV r p-Value
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Landrace
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HSPCB
7 15.26 0.54 3.55 0.518 0.235
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GAPDH
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n geo Mean std dev CV r p-Value
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t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23 t3:24 t3:25 t3:26 t3:27 t3:28 t3:29 t3:30 t3:31 t3:32 t3:33 t3:34
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Table 3 Expression stability analysis of the reference genes by BestKeeper.
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S.-J. Park et al. / Gene xxx (2014) xxx–xxx
et al., 2012). Unfortunately, these RT-qPCR data were normalized using traditionally used reference genes, such as GAPDH, ACTB, and B2M, without choosing appropriate reference genes from previous studies or performing a procedure to select suitable reference genes. However, Larsen et al., used GAPDH for normalization based on previous studies with the expression of the porcine presenilin1 gene, where different reference genes were tested on developing brain tissues (Madsen et al., 2007; Larsen et al., 2012). To obtain accurate quantification data from RT-qPCR experiments, studies on the selection of appropriate reference genes should be performed in various tissue types of multiple pig breeds. Therefore, in this study, we selected appropriate reference genes in four pig breeds. We identified the most suitable reference genes from 15 commonly used candidate genes in the liver, lung, kidney, spleen, stomach, small intestine, and large intestine tissues of Berkshire, Duroc, Landrace, and Yorkshire pigs using the geNorm, NormFinder, and BestKeeper programs. As a result, although the rankings of the candidate reference genes showed slightly different patterns among the programs, the highest ranked genes were similar among the programs. Because the three programs are based on different algorithms and analytical procedures, different suitable reference genes could be selected for each pig species. Therefore, to select the most appropriate and stable reference genes, combination analysis should be performed using the highly ranked reference genes from each program for each pig breed. In the Berkshire pig, three reference genes were selected for optimal number of reference genes. PPIA, TBP, and HSPCB were ranked as the top three genes and showed almost identical stability M values in the geNorm program (Fig. 1). These three genes were also ranked as the top three genes in the NormFinder program and showed almost identical stability values (Table 2). TBP, HSPCB, and PPIA genes were ranked first, third, and fourth, respectively, by the BestKeeper program (Table 3). On the basis of these results, PPIA, TBP, and HSPCB were selected as the most stable and suitable reference genes for Berkshire pigs. In the same way, we
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
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References
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Andersen, C.L., Jensen, J.L., Orntoft, T.F., 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64, 5245–5250. Beekman, L., Tohver, T., Dardari, R., Leguillette, R., 2011. Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease. BMC Mol. Biol. 12, 5. Bruun, C.S., Jensen, L.K., Leifsson, P.S., Nielsen, J., Cirera, S., Jorgensen, C.B., Jensen, H.E., Fredholm, M., 2013. Functional characterization of a porcine emphysema model. Lung 191, 669–675. Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622.
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393
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C
373 374
E
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R
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R
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N C O
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U
363 364
F
401
361 362
O
399 400 Q6
This research was supported by the Priority Research CenteNors Program through the National Research Foundation of Korea (NRF), the Ministry of Education, Science and Technology (2009-0093813), and Export Promotion Technology Development Program, Ministry of Agriculture, Food and Rural Affairs.
359 360
R O
396
358
P
Acknowledgment
356 357
Davoli, R., Gandolfi, G., Braglia, S., Comella, M., Zambonelli, P., Buttazzoni, L., Russo, V., 2011. New SNP of the porcine perilipin 2 (PLIN2) gene, association with carcass traits and expression analysis in skeletal muscle. Mol. Biol. Rep. 38, 1575–1583. Erkens, T., Van Poucke, M., Vandesompele, J., Goossens, K., Van Zeveren, A., Peelman, L.J., 2006. Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A. BMC Biotechnol. 6, 41. Feng, X., Xiong, Y., Qian, H., Lei, M., Xu, D., Ren, Z., 2010. Selection of reference genes for gene expression studies in porcine skeletal muscle using SYBR green qPCR. J. Biotechnol. 150, 288–293. Fontanesi, L., Colombo, M., Tognazzi, L., Scotti, E., Buttazzoni, L., Dall'Olio, S., Davoli, R., Russo, V., 2011. The porcine TBC1D1 gene: mapping, SNP identification, and association study with meat, carcass and production traits in Italian heavy pigs. Mol. Biol. Rep. 38, 1425–1431. Gu, Y.R., Li, M.Z., Zhang, K., Chen, L., Jiang, A.A., Wang, J.Y., Li, X.W., 2011. Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig. J. Anim. Breed. Genet. 128, 319–325. Hasler-Rapacz, J., Prescott, M.F., Von Linden-Reed, J., Rapacz Jr., J.M., Hu, Z., Rapacz, J., 1995. Elevated concentrations of plasma lipids and apolipoproteins B, C-III, and E are associated with the progression of coronary artery disease in familial hypercholesterolemic swine. Arterioscler. Thromb. Vasc. Biol. 15, 583–592. Henriksen, C., Madsen, L.B., Bendixen, C., Larsen, K., 2009. Characterization of the porcine TOR1A gene: the first step towards generation of a pig model for dystonia. Gene 430, 105–115. Huang, J., Ma, G., Zhu, M., Pan, J., Zhang, W., Zhao, S.H., 2012. Molecular characterization of the porcine STAT4 and STAT6 genes. Mol. Biol. Rep. 39, 6959–6965. Kuijk, E.W., du Puy, L., van Tol, H.T., Haagsman, H.P., Colenbrander, B., Roelen, B.A., 2007. Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos. BMC Dev. Biol. 7, 58. Larsen, K., Madsen, L.B., Bendixen, C., 2012. Porcine dorfin: molecular cloning of the RNF19 gene, sequence comparison, mapping and expression analysis. Mol. Biol. Rep. 39, 10053–10062. Li, Q., Domig, K.J., Ettle, T., Windisch, W., Mair, C., Schedle, K., 2011. Evaluation of potential reference genes for relative quantification by RT-qPCR in different porcine tissues derived from feeding studies. Int. J. Mol. Sci. 12, 1727–1734. Li, Q., Tao, Z., Shi, L., Ban, D., Zhang, B., Yang, Y., Zhang, H., Wu, C., 2012. Expression and genome polymorphism of ACSL1 gene in different pig breeds. Mol. Biol. Rep. 39, 8787–8792. Madsen, L.B., Thomsen, B., Larsen, K., Bendixen, C., Holm, I.E., Fredholm, M., Jorgensen, A.L., Nielsen, A.L., 2007. Molecular characterization and temporal expression profiling of presenilins in the developing porcine brain. BMC Neurosci. 8, 72. Maroufi, A., Van Bockstaele, E., De Loose, M., 2010. Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol. Biol. 11, 15. Martino, A., Cabiati, M., Campan, M., Prescimone, T., Minocci, D., Caselli, C., Rossi, A.M., Giannessi, D., Del Ry, S., 2011. Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-alpha mRNA expression. J. Biotechnol. 153, 92–99. McBryan, J., Hamill, R.M., Davey, G., Lawlor, P., Mullen, A.M., 2010. Identification of suitable reference genes for gene expression analysis of pork meat quality and analysis of candidate genes associated with the trait drip loss. Meat Sci. 86, 436–439. McCulloch, R.S., Ashwell, M.S., O'Nan, A.T., Mente, P.L., 2012. Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage. J. Anim. Sci. Biotechnol. 3, 36. Nygard, A.B., Jorgensen, C.B., Cirera, S., Fredholm, M., 2007. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol. Biol. 8, 67. Park, S.J., Huh, J.W., Kim, Y.H., Lee, S.R., Kim, S.H., Kim, S.U., Kim, H.S., Kim, M.K., Chang, K.T., 2013. Selection of internal reference genes for normalization of quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis in the canine brain and other organs. Mol. Biotechnol. 54, 47–57. Pfaffl, M.W., Tichopad, A., Prgomet, C., Neuvians, T.P., 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper– Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–515. Piorkowska, K., Oczkowicz, M., Rozycki, M., Ropka-Molik, K., Piestrzynska-Kajtoch, A., 2011. Novel porcine housekeeping genes for real-time RT-PCR experiments normalization in adipose tissue: assessment of leptin mRNA quantity in different pig breeds. Meat Sci. 87, 191–195. Rozen, S., Skaletsky, H., 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386. Uddin, M.J., Cinar, M.U., Tesfaye, D., Looft, C., Tholen, E., Schellander, K., 2011. Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues. BMC Res. Notes 4, 441. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3 (RESEARCH0034). Wang, S., Li, J., Zhang, A., Liu, M., Zhang, H., 2011. Selection of reference genes for studies of porcine endometrial gene expression on gestational day 12. Biochem. Biophys. Res. Commun. 408, 265–268. Xiang-Hong, J., Yan-Hong, Y., Han-Jin, X., Li-Long, A., Ying-Mei, X., Pei-Rong, J., Ming, L., 2011. Selection of reference genes for gene expression studies in PBMC from Bama miniature pig under heat stress. Vet. Immunol. Immunopathol. 144, 160–166. Xiao, Z., Wang, C., Mo, D., Li, J., Chen, Y., Zhang, Z., Cong, P., 2012. Promoter CpG methylation status in porcine Lyn is associated with its expression levels. Gene 511, 73–78. Yperman, J., De Visscher, G., Holvoet, P., Flameng, W., 2004. Beta-actin cannot be used as a control for gene expression in ovine interstitial cells derived from heart valves. J. Heart Valve Dis. 13, 848–853.
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selected PPIA and TBP as the most stable and suitable reference genes for Duroc pigs; PPIA, TBP, RPL4, and RPS18 as the most stable and suitable reference genes for Landrace pigs; and PPIA, TOP2B, RPL4, and RPS18 as the most stable and suitable reference genes for Yorkshire pigs. Interestingly, PPIA was selected as a suitable reference gene in all pigs. Previous studies showed that PPIA was also selected as a suitable reference gene in different tissues at different ages, articular cartilage, longissimus dorsi, and cardiac tissues (Feng et al., 2010; Martino et al., 2011; Uddin et al., 2011; McCulloch et al., 2012). Therefore, PPIA could be a good reference gene for normalization of RT-qPCR data in the various tissue types of pigs. However, although the PPIA gene was selected in all pigs, different optimal numbers and compositions of reference genes were selected in each pig. SDHA and HPRT1 genes were the least stable according to the geNorm and NormFinder programs. However, these genes were selected as appropriate reference genes in longissimus dorsi muscles of Large White and Meishan pigs, different body tissues, and cardiac tissues (Nygard et al., 2007; Feng et al., 2010; Martino et al., 2011). Therefore, selection of appropriate reference genes for accurate normalization of target genes should be analyzed according to different tissue types, experimental conditions, and pig breeds. Our results also indicated that commonly used traditional reference genes, such as ACTB, GAPDH, and B2M, were not stable or suitable for normalization with gene expression of target genes in the pigs. Therefore, studies on the selection of appropriate reference genes must be performed before quantification of target genes for accurate normalization. In this study, PPIA, TBP, and HSPCB in Berkshire pigs; PPIA, TBP, RPL4, and RPS18 in Landrace pigs; PPIA and TBP in Duroc pigs; and PPIA, TOP2B, RPL4, and RPS18 in Yorkshire pigs were selected as stably expressed and appropriate reference genes for the accurate normalization of RT-qPCR data by the combination analysis of three well-known programs, geNorm, NormFinder, and BestKeeper. The different optimal number and composition of reference genes indicated that studies on the selection of suitable reference genes are essential in RT-qPCR analyses. Also, although the numerous studies have been investigated using crossbred pigs, not a few studies about SNP and expression analysis using various tissues of purebred pigs were reported in recently (Davoli et al., 2011; Fontanesi et al., 2011; Huang et al., 2012; Li et al., 2012). Therefore, these results could provide reliable information for studying various target genes related to diseases, meat quality, litter size, reproduction, and development in pigs, and comparative analysis with purebred and crossbred pigs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.12.052.
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Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
3Q3
Sang-Je Park a,⁎,1, Seul Gi Kwon b,1, Jung Hye Hwang b, Da Hye Park b, Tae Wan Kim b, Chul Wook Kim b,⁎⁎
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Article history: Received 19 August 2014 Received in revised form 22 December 2014 Accepted 25 December 2014 Available online xxxx
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Keywords: Berkshire Duroc Landrace Normalization Reference gene RT-qPCR Yorkshire
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National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungbuk 363-883, Republic of Korea Swine Science and Technology Center, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea
a b s t r a c t
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Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most reliable molecular biology technique for assessment of mRNA expression levels. However, to obtain the accurate RT-qPCR results, the expression levels of genes of interest should be normalized with appropriate reference genes and optimal numbers of reference genes. In this study, we assessed the expression stability of 15 well-known candidate reference genes (ACTB, ALDOA, B2M, GAPDH, HPAR1, HSPCB, PGK1, POLR2G, PPIA, RPL4, RPS18, SDHA, TBP, TOP2B, and YWHAZ) in seven body tissues (liver, lung, kidney, spleen, stomach, small intestine, and large intestine) of Berkshire, Landrace, Duroc, and Yorkshire pigs using three excel-based programs, geNorm, NormFinder, and BestKeeper. Combination analysis of these three programs showed that the stable and appropriate reference genes are PPIA, TBP, and HSPCB in Berkshire pigs; PPIA, TBP, RPL4, and RPS18 in Landrace pigs; PPIA and TBP in Duroc pigs; and PPIA, TOP2B, RPL4, and RPS18 in Yorkshire pigs. Because the four pig breeds had different suitable reference genes, the selection of appropriate reference genes is essential in RT-qPCR analyses. Taken together, our data could help to select reliable reference genes for the normalization of expression levels of various target genes in pigs. © 2014 Published by Elsevier B.V.
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1. Introduction
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Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is one of the most frequently used experimental methods for the evaluation of mRNA expression levels due to its sensitivity, specificity, accuracy, and cost-effectiveness. However, quantification of mRNA levels using RT-qPCR may be affected by various parameters, including RNA quality and purity in RNA extraction steps, differing sample amounts, and enzymatic efficiency in reverse transcription steps, and PCR efficiency (Vandesompele et al., 2002; Park et al., 2013). Therefore, normalization processes should be performed
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Abbreviations: AF, African green monkeys; AS, alternative splicing; BCS1L, BC1 (ubiquinol-cytochrome c reductase) synthesis-like (BCS1L); BO, bonobos; CA, capuchins; CDS, coding sequence; CH, chimpanzees; CO, colobus monkeys; CR, crab-eating monkeys; HERV, human endogenous retrovirus; HU, humans; JA, Japanese monkeys; LA, langurs; LINE, long interspersed element; MA, mandrills; MAR, marmosets; NI, night monkeys; NWM, New World monkey; OWM, Old World monkey; PI, pig-tail monkeys; PPT, polypyrimidine tract; RH, rhesus monkeys; RL, ring-tailed lemurs; SINE, short interspersed element; SP, spider monkeys; SQ, squirrel monkeys; TA, tamarins; TEs, transposable elements; TSS, transcriptional start site; UTR, untranslated region ⁎ Corresponding author. ⁎⁎ Correspondence to: C. W. Kim, Department of Animal Resources Technology, Gyeongnam National University of Science and Technology, Jinju 660-758, Republic of Korea. E-mail addresses: [email protected] (S.-J. Park), [email protected] (C.W. Kim). 1 These authors contributed equally to this work.
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Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs
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using constitutively expressed genes, i.e., reference genes or internal control genes. In previous studies, housekeeping genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), and ribosomal protein s18 (RPS18), were used to normalize mRNA expression without selection of appropriate reference genes. However, these genes have variable expression levels across tissue types, disease state, and different experimental conditions (Vandesompele et al., 2002; Yperman et al., 2004; Maroufi et al., 2010; Beekman et al., 2011). Therefore, to obtain an accurate quantification of target genes, selection of suitable and stable reference genes among the various candidate reference genes should be performed according to breed, tissue and cell type, and experimental conditions. The pig (Sus scrofa) is an economically important livestock animal for meat production. It is also an ideal animal model for the study of human disease due to its similar genome size and organization (Hasler-Rapacz et al., 1995; Uddin et al., 2011). Therefore, numerous quantitative analyses of target genes have been performed using RT-qPCR experiments in various pig breeds to improve meat quality and to study human diseases (Henriksen et al., 2009; Li et al., 2012; Xiao et al., 2012; Bruun et al., 2013). To our knowledge, several reference gene studies have been performed in various pig tissue samples, including fat-type tissues, muscle-type tissues, articular cartilage, backfat tissues, longissimus dorsi muscle, peripheral blood mononuclear cells (PBMC), different embryonic stage samples, epithelial endometria, cardiac tissue, and others in various purebred and hybrid pig breeds
http://dx.doi.org/10.1016/j.gene.2014.12.052 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
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2.1. Ethics statement
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The Animal Care and Use Committee of GNTECH (Gyeongnam National University of Science and Technology) specifically waived the need for consent because approval by the ethics committee is not required for the slaughter of farm animals in the Republic of Korea. Used farm pigs were generated from purebred breeding pigs and these pigs were tested and regulated at the First Korea Swine Testing Station. All pigs were slaughtered in a commercial abattoir (Bukyung livestock joint market). Slaughterhouse management gave the necessary permissions for the tissue collection. Pigs used in this study were slaughtered in accordance with the guidelines on animal care and use established by the Republic of Korea's Animal Protection Act and related laws. Four Pig breeds weighing approximately 110 kg (average of age 180 days) were transported to an abattoir near the experimental station. They were slaughtered by stunning with electrical tongs (300 V for 3 s) after 12 h of feed restriction. The shocked pigs were exsanguinated while being hanged.
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2.2. Total RNA isolation and cDNA preparation
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After exsanguination, seven different body tissues (liver, lung, kidney, spleen, stomach, small intestine, and large intestine) of Berkshire (n = 3, male), Landrace (n = 3, male), Duroc (n = 3, male), and Yorkshire (n = 3, male) pigs were directly collected, and all samples were immediately snap-frozen and stored in liquid nitrogen, prior to total RNA extraction. In this study, we investigated selection of reference genes using three male pigs in each breeds. Because, almost slaughtered purebred pigs were male pigs, and female purebred pigs were used for breeding in Korea farm. Therefore, we first analyzed using male pig breeds. Total RNA was extracted from seven different body tissues using the RNeasy Plus Mini kit (Qiagen) following the manufacturer's instructions. In this kit, a gDNA Eliminator spin column was used to remove DNA contamination from the total RNA preparations. Total RNA was quantified using a NanoDrop® ND-1000 UV–vis Spectrophotometer. Reverse transcription was performed using SuperScript™ II Reverse Transcriptase (Invitrogen) with RNase OUT (Invitrogen) following the manufacturer's instructions. To remove RNA complementary to the cDNA, RNase H (Invitrogen) was used. We performed PCR amplification without the reverse transcription (RT) reaction using pure RNA
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Specific primer pairs for candidate reference genes were designed using the Primer3 program (http://frodo.wi.mit.edu/primer3/; Table 1) (Rozen and Skaletsky, 2000). BLAST searches were performed to confirm gene specificity of the primer sequences, and the results showed the absence of multi-locus matching at individual primer sites. Most primers spanned at least 2 exons or had a large intron sequence between the forward and reverse primers to avoid falsepositive amplification of contaminating genomic DNA in the RNA samples. The nucleotide sequences of the RT-PCR products for the 15 candidate reference genes were obtained using standard molecular cloning and sequencing procedures (Fig. S1). Amplification efficiencies and correlation coefficients (R2 values) of the 15 genes were generated using the slopes of the standard curves obtained from serial dilutions. Standard curves from a 10-fold dilution series were used to calculate the amplification efficiency (Table 1). The amplification efficiency was calculated according to the formula: efficiency (%) = (10(−1/slope) − 1) × 100. The efficiency range of the real-time RT-PCR amplifications for all the tested genes was 86% to 106%.
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Real-time RT-PCR using SYBR Green was performed using a Rotor Gene-Q thermocycler (Qiagen). In each run, 1 μL of cDNA was used as the template for each reaction. The samples were added to 19 μL of the reaction mixture (7 μL H2O, 10 μL Rotor Gene SYBR Green PCR mastermix (Qiagen), and 1 μL each of the forward and reverse primers). Real-time RT-PCR amplification of the 15 genes was performed in 40 cycles of 94 °C for 5 s and 60 °C for 10 s. The amplification specificity of each RT-qPCR assay was confirmed by melting curve analysis. The temperature range for the analysis of the melting curves was 70 °C–95 °C for 5 s. As shown in Fig. S2, each primer pair showed a single, sharp peak, indicating that the primers amplified a single, specific PCR product. Amplification was not detected in the no-template controls (NTC) for all candidate reference genes (Table 1). These experiments were performed at least three times.
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2.5. Characterization of the analysis programs
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The geNorm program (Vandesompele et al., 2002) provides a measure of gene expression stability (M value), which is the mean pair-wise variation between an individual gene and all other tested control genes. This method differs from model-based approaches because it compares genes based on the similarity of their expression profiles. Cq values are converted to scale expression quantities using the ΔCq method and are recorded in the geNorm program, which then ranks the genes based on their M values; the genes with high M values are less stably expressed and would make bad reference genes and those with low M values are stably expressed and would make good reference genes. In addition, the geNorm program can calculate the optimal number of required reference genes for obtaining reliable results from RT-qPCR studies. This calculation was performed by analysis of the pair-wise variation (V value) of sequential normalization factors (NF) with an increasing number of reference genes (NFn and NFn + 1). NormFinder (Andersen et al., 2004) is a tool to identify optimally stable reference genes through the determination of expression stabilities using a model-based approach in Microsoft Excel. This program focuses on finding the two genes with the least intra- and inter-group expression variation or the most stable reference gene in intra-group expression variation. In this program, genes with lower stability values
171
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98 99
C
92 93
E
90 91
R
88 89
R
86 87
O
84 85
C
82 83
N
80 81
U
79
2.3. Primer design and standard curve analysis
F
2. Materials and methods
77 78
O
95
75 76
samples (no-RT control) and determined that the prepared mRNA 133 samples did not contain genomic DNA. 134
P
94
(Erkens et al., 2006; Kuijk et al., 2007; Nygard et al., 2007; McBryan et al., 2010; Gu et al., 2011; Li et al., 2011; Martino et al., 2011; Piorkowska et al., 2011; Uddin et al., 2011; Wang et al., 2011; Xiang-Hong et al., 2011; McCulloch et al., 2012). However, studies on the selection of appropriate reference genes have not yet been performed on body tissues of Berkshire, Landrace, Duroc, and Yorkshire pigs. Therefore, we selected suitable reference genes and performed a comparative analysis in these four pig breeds. The aim of this study was to select and evaluate the stability of fifteen candidate reference genes using seven body tissues of Berkshire, Landrace, Duroc, and Yorkshire pigs. The stabilities of ACTB, aldolase A, fructose-bisphosphate (ALDOA), β-2-microglobulin (B2M), GAPDH, hypoxanthine phosphoribosyltransferase 1 (HPRT1), heat shock 90 kDa protein 1, beta (HSPCB), phosphoglycerate kinase 1 (PGK1), peptidylprolyl isomerase A (cyclophilin A) (PPIA), polymerase (RNA) II (DNA directed) polypeptide G (POLR2G), ribosomal protein L4 (RPL4), RPS18, succinate dehydrogenase complex subunit A (SDHA), TATA box binding protein (TPB), topoisomerase II beta (TOP2B), and tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein ζ polypeptide (YWHAZ) were analyzed using the geNorm (Vandesompele et al., 2002), NormFinder (Andersen et al., 2004), and BestKeeper (Pfaffl et al., 2004) programs.
E
73 74
S.-J. Park et al. / Gene xxx (2014) xxx–xxx
R O
2
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
157 158 159 160 161 162 163 164 165 166 167 168 169
172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192
t1:3
N
Table 1 Primers for the 15 candidate reference genes and parameters derived from RT-qPCR data analysis. Gene name
ACTB
Beta actin
ALDOA
Aldolase A, fructose-bisphosphate
B2M
Beta-2-microglobulin
GAPDH
Glyceraldehyde-3-phosphate dehydrogenase
NM_001206359.1
HPRT1
Hypoxanthine phosphoribosyltransferase 1
Martino et al. (2011)
HSPCB
Heat shock 90 kDa protein 1, beta
Gu et al. (2011)
PGK1
Phosphoglycerate kinase 1
Wang et al. (2011)
POLR2G
Polymerase (RNA) II (DNA directed) polypeptide G
XM_005660782.1
PPIA
Peptidylprolyl isomerase A (cyclophilin A)
Uddin et al. (2011)
RPL4
Ribosomal protein L4
Uddin et al. (2011)
RPS18
RIBOSOMAL protein S18
NM_213940.1
SDHA
Succinate dehydrogenase complex, subunit A
Erkens et al. (2006)
TBP
TATA box binding protein
Martino et al. (2011)
TOP2B
Topoisomerase II beta
NM_001258386.1
YWHAZ
Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide
Erkens et al. (2006)
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29 t1:30 t1:31 t1:32 t1:33 t1:34 t1:35 t1:36 t1:37
C
Abbreviation
GenBank accession no. or reference
O
N.d.: Not detected. a If a primer is located on 2 exons, the junctions are shown with a virgule. b No template control.
Primera
Exon(s)
Amplicon size (bp)
PCR efficiency (%)
R2
NTCb (Cq)
3rd 3rd/4th Unknown
114
106
0.99761
N.d.
142
99
0.99933
N.d.
1st/2nd 3rd 4th 5th/6th 1st 2nd 8th 9th 2nd/3rd 3rd 4th/5th 7th 4th 5th 5th 6th/7th 2nd/3rd 4th/5th 5th 6th Unknown
166
105
0.99209
N.d.
130
102
0.99271
N.d.
181
93
0.99562
N.d.
131
98
0.9931
N.d.
126
96
0.99957
N.d.
181
99
0.99831
N.d.
171
98
0.9982
N.d.
185
99
0.9833
N.d.
74
96
0.99792
N.d.
193
86
0.99861
N.d.
124
88
0.9975
N.d.
16th/17th 18th 4th 5th
115
94
0.99526
N.d.
97
0.99555
N.d.
Forward (F)/reverse (R) Martino et al. (2011)
R
Gu et al. (2011)
R
Martino et al. (2011)
E
C
F: TCTGGCACCACACCTTCT R: TGAT/CTGGGTCATCTTCTCAC F: GAACCAACGGCGAGACAA R: ATGATGGCGAGGGAGGAG F: TTCA/CACCGCTCCAGTAG R: CCAGATACATAGCAGTTCAGG F: ATCCTGGGCTACACTGAGGA R: TGTCGTAC/CAGGAAATGAGCT F: CCGAGGATTTGGAAAAGGT R: CTATTTCTGTTCAGTGCTTTGATGT F: GGCAGAAGACAAGGAGAAC R: CAGACTGGGAGGTATGGTAG F: AGATAACGAACAACCAGAG/G R: TGTCAGGCATAGGGATACC F: CTCAAGTCAACAAG/GTCGGAC R: GTCCCAACAATCTTCAGGCG F: CACAAACGGTTCCCAGTTTT R: TGTCCACAGTCAGCAATGGT F: AGGAGGCTGTTCTGCTTCTG R: TC/CAGGGATGTTTCTGAAGG F: GCGATTAAG/GGTGTAGGACG R: GAC/CTGGCTGTACTTCCCAT F: GAACCGAAGATGGCAAGA R: CAGGAGATCCAAGGCAAA F: GATGGACGTTCGGTTTAGG R: AGCAGCACAGTACGAGCAA F: AA/GGGCGAGAGGTCAATGAT R: ACATCTTCTCGTTCTTGCGC F: ATGCAACCAACACATCCTATC R: GCATTATTAGCGTGCTGTCTT
T
E
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S.-J. Park et al. / Gene xxx (2014) xxx–xxx
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
t1:1 t1:2
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3
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3. Results
209
3.1. Selection of candidate reference genes
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To select suitable reference genes for the seven body tissues of four pig breeds, we chose 15 candidate reference genes based on previous studies in which commonly used reference genes were determined to be stably expressed. We also chose genes belonging to different functional classes to minimize the risk of coregulation. On the basis of these criteria, our candidate genes were ACTB, ALDOA, B2M, GAPDH, HPRT1, HSPCB, PGK1, PPIA, POLR2G, RPL4, RPS18, SDHA, TBP, TOP2B, and YWHAZ (Table 1) (Erkens et al., 2006; Kuijk et al., 2007; Nygard et al.,
C E R R O
217
C
215 216
N
213 214
221
U
211 212
a) GeNorm analysis The stability values (M value) of the 15 candidate reference genes were calculated using the geNorm program (Fig. 1). PPIA and TBP were identified as the two most stable genes in Berkshire and Duroc pigs, with low M-values of 0.21 and 0.15, respectively. RPL4 and RPS18 had low M-values of 0.16 and 0.18 in Landrace and Yorkshire pigs, respectively, and were identified as the two most stable genes. In Berkshire pigs, the most stable genes following PPIA were HSPCB (0.23), RPS18 (0.24), RPL4 (0.27), POLR2G (0.36), B2M (0.43), PGK1 (0.50), TOP2B (0.56), GAPDH (0.61), YWHAZ (0.68), ALDOA (0.73), ACTB (0.76), SDHA (0.84), and HPRT1 (0.97). In Duroc pigs, the most stable genes following TBP were RPL4 (0.25), HSPCB (0.36), RPS18 (0.41), TOP2B (0.46), B2M (0.50), ALDOA (0.53), POLR2G (0.58), GAPDH (0.61), ACTB (0.66), YWHAZ (0.69), PGK1 (0.72), SDHA (0.80), and HPRT1 (0.90). In Landrace pigs, the most stable genes following RPL4 were PPIA (0.28), TOP2B (0.35), TBP (0.38), HSPCB (0.41), ALDOA (0.46), ACTB (0.50), YWHAZ (0.53), GAPDH (0.57), B2M (0.61), POLR2G (0.65), PGK1 (0.69), SDHA (0.76), and HPRT1 (0.87). In Yorkshire pigs, the most stable genes following RPS18 were PPIA (0.30), TOP2B (0.33), HSPCB (0.38), POLR2G (0.42), TBP (0.45), B2M (0.48), ACTB (0.52), ALDOA (0.56), YWHAZ (0.59), GAPDH (0.63), PGK1 (0.66), SDHA (0.75), and HPRT1 (0.87). According to Vandesompele et al. (the developers of the geNorm program), the use of a minimal number of the most stable reference genes is recommended for the calculation of the normalization
F
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All experiments were performed according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines (Bustin et al., 2009).
200 201
220
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205 206
199
3.2. Expression stability of candidate reference genes
R O
2.6. MIQE guidelines
197 198
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204
195 196
2007; Gu et al., 2011; Li et al., 2011; Martino et al., 2011; Uddin et al., 218 2011; Wang et al., 2011; McCulloch et al., 2012). 219
T
202 203
show less varied expressions and have a stably expressed pattern, and genes with higher stability values show more varied expressions and have the least stably expressed pattern. BestKeeper (Pfaffl et al., 2004) index is created using the geometric mean of each candidate gene's Cq values. This program determines the most stably expressed genes based on correlation coefficient (r) analysis for all pairs of candidate reference genes (≤10 genes) and calculates the percentage coefficient of variation (CV) and standard deviation (SD) using each candidate gene's crossing point (CP) value (the quantification cycle value; Cq). In this program, genes with higher r values and lower CV and SD values are stable reference genes.
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S.-J. Park et al. / Gene xxx (2014) xxx–xxx
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4
Fig. 1. Ranking of 15 candidate reference genes using the geNorm program. The average expression stability (M) of 15 candidate reference genes and the best combination of two genes were calculated for Berkshire pigs (A), Duroc pigs (B), Landrace pigs (C), and Yorkshire pigs (D). Lower M values indicate more stable expression.
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
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t2:1 t2:2
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factor (NF) (Vandesompele et al., 2002). Therefore, we analyzed the optimal number of required reference genes for obtaining reliable results from RT-qPCR studies (Fig. 2). The original paper using the geNorm program proposed 0.15 as the cut-off value, which suggests that additional reference genes would be unnecessary if the V value was b0.15. The pair-wise variation V2/3 was 0.070 in Berkshire pigs, 0.098 in Duroc pigs, 0.110 in Landrace pigs, and 0.116 in Yorkshire pigs. All values are lower than 0.15; however, the V value decreased significantly with the addition of reference genes, except for Duroc pigs. Therefore, to calculate the NF more accurately, the two most stable reference genes were recommended for Duroc pigs, the
N C O
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C
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Fig. 2. Determination of the optimal number of reference genes for normalization. The geNorm program calculated the normalization factor (NF) from at least two genes and the variable V defines the pair-wise variation between two sequential NF values. Berkshire pigs (A), Duroc pigs (B), Landrace pigs (C), and Yorkshire pigs (D).
Table 2 Gene stability value calculations by NormFinder.
t2:3
Berkshire
t2:4 t2:5
Gene name
Stability value
Gene name
Stability value
Gene name
Stability value
Gene name
Stability value
t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20
PPIA TBP HSPCB RPS18 RPL4 TOP2B GAPDH POLR2G PGK1 B2M ALDOA YWHAZ ACTB SDHA HPRT1
0.062 0.073 0.073 0.106 0.161 0.393 0.395 0.41 0.447 0.467 0.618 0.643 0.67 0.811 1.212
PPIA TBP RPL4 HSPCB TOP2B GAPDH POLR2G RPS18 B2M ALDOA PGK1 ACTB YWHAZ SDHA HPRT1
0.078 0.079 0.131 0.297 0.305 0.341 0.377 0.381 0.403 0.424 0.454 0.574 0.615 0.822 1.002
PPIA TBP RPL4 RPS18 TOP2B GAPDH HSPCB POLR2G ALDOA B2M PGK1 ACTB YWHAZ SDHA HPRT1
0.111 0.134 0.156 0.174 0.25 0.288 0.369 0.393 0.404 0.476 0.494 0.501 0.557 0.718 1.054
PPIA TOP2B POLR2G RPS18 RPL4 TBP HSPCB GAPDH B2M PGK1 ACTB ALDOA YWHAZ SDHA HPRT1
0.046 0.176 0.201 0.204 0.218 0.25 0.289 0.356 0.387 0.411 0.475 0.495 0.573 0.824 1.11
U
Duroc
Landrace
Yorkshire
three most stable reference genes were recommended for Berkshire and Yorkshire pigs, and the four most stable reference genes were recommended for Landrace pigs as the optimal numbers of reference genes. b) NormFinder analysis NormFinder calculates the stability value and standard error according to the expression variation of the candidate reference genes. Analysis of our NormFinder data showed that PPIA was the most stable reference gene with the lowest stability value in all pig breeds (Table 2). TBP (0.073), HSPCB (0.073), RPS18 (0.106), RPL4 (0.161), TOP2B (0.393), GAPDH (0.395), POLR2G (0.410), PGK1 (0.447), B2M (0.467), ALDOA (0.618), YWHAZ (0.643), ACTB (0.670), SDHA (0.811), and HPRT1 (1.212) had increasing stability values in Berkshire pigs; TBP (0.079), RPL4 (0.131), HSPCB (0.297), TOP2B (0.305), GAPDH (0.341), POLR2G (0.377), RPS18 (0.381), B2M (0.403), ALDOA (0.424), PGK1 (0.454), ACTB (0.574), YWHAZ (0.615), SDHA (0.822), and HPRT1 (1.002) had increasing stability values in Duroc pigs; TBP (0.134), RPL4 (0.156), RPS18 (0.174), TOP2B (0.250), GAPDH (0.288), HSPCB (0.369), POLR2G (0.393), ALDOA (0.404), B2M (0.476), PGK1 (0.494), ACTB (0.501), YWHAZ (0.557), SDHA (0.718), and HPRT1 (1.054) had increasing stability values in Landrace pigs; and TOP2B (0.176), POLR2G (0.201), RPS18 (0.204), RPL4 (0.218), TBP (0.250), HSPCB (0.289), GAPDH (0.356), B2M (0.387), PGK1 (0.411), ACTB (0.475), ALDOA (0.495), YWHAZ (0.573), SDHA (0.824), and HPRT1 (1.110) had increasing stability values in Yorkshire pigs. c) BestKeeper analysis The BestKeeper program is also an Excel-based software tool. On the basis of the correlation coefficients (r), CV, and SD values, the optimal reference gene was determined (Table 3). This program can calculate r values for up to 10 genes. Therefore, we selected
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288
6
Berkshire
t3:4
295 296 297 298 299 300 301 302
PGK1
POLR2G
PPIA
RPL4
RPS18
TBP
TOP2B
7 16.05 0.55 3.45 0.966 0.001
7 17.58 0.74 4.22 0.837 0.019
7 18.30 0.39 2.13 0.616 0.141
7 14.36 0.50 3.50 0.993 0.001
7 14.26 0.45 3.18 0.946 0.001
7 13.61 0.69 5.06 0.986 0.001
7 20.90 0.53 2.52 0.966 0.001
7 19.54 0.79 4.02 0.854 0.014
ALDOA 16.24 0.83 5.13 0.851 0.015
B2M 13.12 0.76 5.80 0.861 0.013
GAPDH 15.62 0.73 4.67 0.805 0.029
HSPCB 16.03 0.66 4.13 0.911 0.004
POLR2G 18.20 0.49 2.71 0.679 0.093
PPIA 15.15 0.56 3.66 0.955 0.001
RPL4 14.26 0.45 3.17 0.992 0.001
RPS18 13.65 0.39 2.82 0.804 0.029
TBP 20.91 0.65 3.11 0.948 0.001
TOP2B 19.61 0.94 4.77 0.945 0.001
ACTB 15.15 0.73 4.79 0.974 0.001
ALDOA 16.34 0.58 3.52 0.948 0.001
GAPDH 15.31 0.38 2.51 0.349 0.444
HSPCB 15.75 0.43 2.72 0.815 0.026
PPIA 14.63 0.40 2.74 0.842 0.017
RPL4 14.15 0.27 1.90 0.914 0.004
RPS18 13.69 0.30 2.18 0.933 0.002
TBP 21.21 0.52 2.46 0.815 0.026
TOP2B 19.58 0.52 2.63 0.853 0.015
YWHAZ 17.30 0.70 4.06 0.930 0.002
ACTB 15.15 0.73 4.79 0.981 0.001
ALDOA 16.13 0.57 3.54 0.832 0.020
B2M 13.91 0.66 4.75 0.820 0.024
HSPCB 15.80 0.45 2.86 0.923 0.003
POLR2G 18.60 0.24 1.29 0.396 0.381
PPIA 14.64 0.31 2.11 0.840 0.018
RPS18 13.78 0.25 1.79 0.798 0.032
TBP 21.11 0.58 2.76 0.873 0.010
TOP2B 19.20 0.44 2.31 0.958 0.001
geo Mean std dev CV r p-Value Yorkshire
T
n number of samples, geo Mean the geometric mean of Cq, std dev standard deviation of Cq, CV coefficient of variation, and r coefficient of correlation. High ranked genes were described in bold.
C
the 10 best candidate genes using the results of the geNorm and NormFinder programs. In the BestKeeper program, genes with higher r value (≥0.900) and lower CV and SD values are stable and suitable reference genes. In Berkshire pigs, the POLR2G gene had the lowest CV (2.13) and SD (0.39) values among the 10 candidate reference genes, and was stably expressed across all tested samples. However, PORL2G had a low r value (0.616) with the other candidate reference genes, which suggests that its expression was not correlated with the expression patterns of the other candidate reference genes. Therefore, we selected TBP as the reference gene for Berkshire pigs because it was stably expressed with high r value (0.966) and low CV (2.52) and SD (0.53) values. TBP, RPL4, and TOP2B were selected as stable reference genes for Duroc, Landrace, and Yorkshire pigs, respectively.
N
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4. Discussion
305
Gene expression analysis is the one of the most commonly and widely used strategies in molecular biology studies, and RT-qPCR has become the most effective method for quantifying gene expression due to its accuracy, specificity, and cost-effectiveness. However, RT-qPCR data can be influenced by normalization with different reference genes. Therefore, the selection of appropriate reference genes is essential for the reliable and reproducible assessment of gene expression. Indeed, many studies on the selection of suitable reference genes have been performed in various species, in different cell or tissue types, and under different experimental conditions. Currently, to understand the gene function and the associations between various genes, meat quality, and reproduction, quantitative analyses have been performed using RT-qPCR in different body tissues and embryos of pigs (Davoli et al., 2011; Huang et al., 2012; Larsen et al., 2012; Li
308 309 310 311 312 313 314 315 316 317 318
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304
306 307
RPL4 14.41 0.23 1.62 0.845 0.017
E
geo Mean std dev CV r p-Value
R O
Landrace
O
geo Mean std dev CV r p-Value
F
Duroc
E
293 294
HSPCB
7 15.26 0.54 3.55 0.518 0.235
R
291 292
GAPDH
7 13.35 0.69 5.13 0.759 0.048
R
289 290
B2M
O
t3:35 t3:36
n geo Mean std dev CV r p-Value
C
t3:5 t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23 t3:24 t3:25 t3:26 t3:27 t3:28 t3:29 t3:30 t3:31 t3:32 t3:33 t3:34
P
t3:3
Table 3 Expression stability analysis of the reference genes by BestKeeper.
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t3:1 t3:2 Q1
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et al., 2012). Unfortunately, these RT-qPCR data were normalized using traditionally used reference genes, such as GAPDH, ACTB, and B2M, without choosing appropriate reference genes from previous studies or performing a procedure to select suitable reference genes. However, Larsen et al., used GAPDH for normalization based on previous studies with the expression of the porcine presenilin1 gene, where different reference genes were tested on developing brain tissues (Madsen et al., 2007; Larsen et al., 2012). To obtain accurate quantification data from RT-qPCR experiments, studies on the selection of appropriate reference genes should be performed in various tissue types of multiple pig breeds. Therefore, in this study, we selected appropriate reference genes in four pig breeds. We identified the most suitable reference genes from 15 commonly used candidate genes in the liver, lung, kidney, spleen, stomach, small intestine, and large intestine tissues of Berkshire, Duroc, Landrace, and Yorkshire pigs using the geNorm, NormFinder, and BestKeeper programs. As a result, although the rankings of the candidate reference genes showed slightly different patterns among the programs, the highest ranked genes were similar among the programs. Because the three programs are based on different algorithms and analytical procedures, different suitable reference genes could be selected for each pig species. Therefore, to select the most appropriate and stable reference genes, combination analysis should be performed using the highly ranked reference genes from each program for each pig breed. In the Berkshire pig, three reference genes were selected for optimal number of reference genes. PPIA, TBP, and HSPCB were ranked as the top three genes and showed almost identical stability M values in the geNorm program (Fig. 1). These three genes were also ranked as the top three genes in the NormFinder program and showed almost identical stability values (Table 2). TBP, HSPCB, and PPIA genes were ranked first, third, and fourth, respectively, by the BestKeeper program (Table 3). On the basis of these results, PPIA, TBP, and HSPCB were selected as the most stable and suitable reference genes for Berkshire pigs. In the same way, we
Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
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References
402 403 404 405 406 407 408 409 410 411 412 413 414 415
Andersen, C.L., Jensen, J.L., Orntoft, T.F., 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 64, 5245–5250. Beekman, L., Tohver, T., Dardari, R., Leguillette, R., 2011. Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease. BMC Mol. Biol. 12, 5. Bruun, C.S., Jensen, L.K., Leifsson, P.S., Nielsen, J., Cirera, S., Jorgensen, C.B., Jensen, H.E., Fredholm, M., 2013. Functional characterization of a porcine emphysema model. Lung 191, 669–675. Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L., Vandesompele, J., Wittwer, C.T., 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622.
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393
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This research was supported by the Priority Research CenteNors Program through the National Research Foundation of Korea (NRF), the Ministry of Education, Science and Technology (2009-0093813), and Export Promotion Technology Development Program, Ministry of Agriculture, Food and Rural Affairs.
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Acknowledgment
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Davoli, R., Gandolfi, G., Braglia, S., Comella, M., Zambonelli, P., Buttazzoni, L., Russo, V., 2011. New SNP of the porcine perilipin 2 (PLIN2) gene, association with carcass traits and expression analysis in skeletal muscle. Mol. Biol. Rep. 38, 1575–1583. Erkens, T., Van Poucke, M., Vandesompele, J., Goossens, K., Van Zeveren, A., Peelman, L.J., 2006. Development of a new set of reference genes for normalization of real-time RT-PCR data of porcine backfat and longissimus dorsi muscle, and evaluation with PPARGC1A. BMC Biotechnol. 6, 41. Feng, X., Xiong, Y., Qian, H., Lei, M., Xu, D., Ren, Z., 2010. Selection of reference genes for gene expression studies in porcine skeletal muscle using SYBR green qPCR. J. Biotechnol. 150, 288–293. Fontanesi, L., Colombo, M., Tognazzi, L., Scotti, E., Buttazzoni, L., Dall'Olio, S., Davoli, R., Russo, V., 2011. The porcine TBC1D1 gene: mapping, SNP identification, and association study with meat, carcass and production traits in Italian heavy pigs. Mol. Biol. Rep. 38, 1425–1431. Gu, Y.R., Li, M.Z., Zhang, K., Chen, L., Jiang, A.A., Wang, J.Y., Li, X.W., 2011. Evaluation of endogenous control genes for gene expression studies across multiple tissues and in the specific sets of fat- and muscle-type samples of the pig. J. Anim. Breed. Genet. 128, 319–325. Hasler-Rapacz, J., Prescott, M.F., Von Linden-Reed, J., Rapacz Jr., J.M., Hu, Z., Rapacz, J., 1995. Elevated concentrations of plasma lipids and apolipoproteins B, C-III, and E are associated with the progression of coronary artery disease in familial hypercholesterolemic swine. Arterioscler. Thromb. Vasc. Biol. 15, 583–592. Henriksen, C., Madsen, L.B., Bendixen, C., Larsen, K., 2009. Characterization of the porcine TOR1A gene: the first step towards generation of a pig model for dystonia. Gene 430, 105–115. Huang, J., Ma, G., Zhu, M., Pan, J., Zhang, W., Zhao, S.H., 2012. Molecular characterization of the porcine STAT4 and STAT6 genes. Mol. Biol. Rep. 39, 6959–6965. Kuijk, E.W., du Puy, L., van Tol, H.T., Haagsman, H.P., Colenbrander, B., Roelen, B.A., 2007. Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos. BMC Dev. Biol. 7, 58. Larsen, K., Madsen, L.B., Bendixen, C., 2012. Porcine dorfin: molecular cloning of the RNF19 gene, sequence comparison, mapping and expression analysis. Mol. Biol. Rep. 39, 10053–10062. Li, Q., Domig, K.J., Ettle, T., Windisch, W., Mair, C., Schedle, K., 2011. Evaluation of potential reference genes for relative quantification by RT-qPCR in different porcine tissues derived from feeding studies. Int. J. Mol. Sci. 12, 1727–1734. Li, Q., Tao, Z., Shi, L., Ban, D., Zhang, B., Yang, Y., Zhang, H., Wu, C., 2012. Expression and genome polymorphism of ACSL1 gene in different pig breeds. Mol. Biol. Rep. 39, 8787–8792. Madsen, L.B., Thomsen, B., Larsen, K., Bendixen, C., Holm, I.E., Fredholm, M., Jorgensen, A.L., Nielsen, A.L., 2007. Molecular characterization and temporal expression profiling of presenilins in the developing porcine brain. BMC Neurosci. 8, 72. Maroufi, A., Van Bockstaele, E., De Loose, M., 2010. Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol. Biol. 11, 15. Martino, A., Cabiati, M., Campan, M., Prescimone, T., Minocci, D., Caselli, C., Rossi, A.M., Giannessi, D., Del Ry, S., 2011. Selection of reference genes for normalization of real-time PCR data in minipig heart failure model and evaluation of TNF-alpha mRNA expression. J. Biotechnol. 153, 92–99. McBryan, J., Hamill, R.M., Davey, G., Lawlor, P., Mullen, A.M., 2010. Identification of suitable reference genes for gene expression analysis of pork meat quality and analysis of candidate genes associated with the trait drip loss. Meat Sci. 86, 436–439. McCulloch, R.S., Ashwell, M.S., O'Nan, A.T., Mente, P.L., 2012. Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage. J. Anim. Sci. Biotechnol. 3, 36. Nygard, A.B., Jorgensen, C.B., Cirera, S., Fredholm, M., 2007. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol. Biol. 8, 67. Park, S.J., Huh, J.W., Kim, Y.H., Lee, S.R., Kim, S.H., Kim, S.U., Kim, H.S., Kim, M.K., Chang, K.T., 2013. Selection of internal reference genes for normalization of quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis in the canine brain and other organs. Mol. Biotechnol. 54, 47–57. Pfaffl, M.W., Tichopad, A., Prgomet, C., Neuvians, T.P., 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper– Excel-based tool using pair-wise correlations. Biotechnol. Lett. 26, 509–515. Piorkowska, K., Oczkowicz, M., Rozycki, M., Ropka-Molik, K., Piestrzynska-Kajtoch, A., 2011. Novel porcine housekeeping genes for real-time RT-PCR experiments normalization in adipose tissue: assessment of leptin mRNA quantity in different pig breeds. Meat Sci. 87, 191–195. Rozen, S., Skaletsky, H., 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386. Uddin, M.J., Cinar, M.U., Tesfaye, D., Looft, C., Tholen, E., Schellander, K., 2011. Age-related changes in relative expression stability of commonly used housekeeping genes in selected porcine tissues. BMC Res. Notes 4, 441. Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., Speleman, F., 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3 (RESEARCH0034). Wang, S., Li, J., Zhang, A., Liu, M., Zhang, H., 2011. Selection of reference genes for studies of porcine endometrial gene expression on gestational day 12. Biochem. Biophys. Res. Commun. 408, 265–268. Xiang-Hong, J., Yan-Hong, Y., Han-Jin, X., Li-Long, A., Ying-Mei, X., Pei-Rong, J., Ming, L., 2011. Selection of reference genes for gene expression studies in PBMC from Bama miniature pig under heat stress. Vet. Immunol. Immunopathol. 144, 160–166. Xiao, Z., Wang, C., Mo, D., Li, J., Chen, Y., Zhang, Z., Cong, P., 2012. Promoter CpG methylation status in porcine Lyn is associated with its expression levels. Gene 511, 73–78. Yperman, J., De Visscher, G., Holvoet, P., Flameng, W., 2004. Beta-actin cannot be used as a control for gene expression in ovine interstitial cells derived from heart valves. J. Heart Valve Dis. 13, 848–853.
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selected PPIA and TBP as the most stable and suitable reference genes for Duroc pigs; PPIA, TBP, RPL4, and RPS18 as the most stable and suitable reference genes for Landrace pigs; and PPIA, TOP2B, RPL4, and RPS18 as the most stable and suitable reference genes for Yorkshire pigs. Interestingly, PPIA was selected as a suitable reference gene in all pigs. Previous studies showed that PPIA was also selected as a suitable reference gene in different tissues at different ages, articular cartilage, longissimus dorsi, and cardiac tissues (Feng et al., 2010; Martino et al., 2011; Uddin et al., 2011; McCulloch et al., 2012). Therefore, PPIA could be a good reference gene for normalization of RT-qPCR data in the various tissue types of pigs. However, although the PPIA gene was selected in all pigs, different optimal numbers and compositions of reference genes were selected in each pig. SDHA and HPRT1 genes were the least stable according to the geNorm and NormFinder programs. However, these genes were selected as appropriate reference genes in longissimus dorsi muscles of Large White and Meishan pigs, different body tissues, and cardiac tissues (Nygard et al., 2007; Feng et al., 2010; Martino et al., 2011). Therefore, selection of appropriate reference genes for accurate normalization of target genes should be analyzed according to different tissue types, experimental conditions, and pig breeds. Our results also indicated that commonly used traditional reference genes, such as ACTB, GAPDH, and B2M, were not stable or suitable for normalization with gene expression of target genes in the pigs. Therefore, studies on the selection of appropriate reference genes must be performed before quantification of target genes for accurate normalization. In this study, PPIA, TBP, and HSPCB in Berkshire pigs; PPIA, TBP, RPL4, and RPS18 in Landrace pigs; PPIA and TBP in Duroc pigs; and PPIA, TOP2B, RPL4, and RPS18 in Yorkshire pigs were selected as stably expressed and appropriate reference genes for the accurate normalization of RT-qPCR data by the combination analysis of three well-known programs, geNorm, NormFinder, and BestKeeper. The different optimal number and composition of reference genes indicated that studies on the selection of suitable reference genes are essential in RT-qPCR analyses. Also, although the numerous studies have been investigated using crossbred pigs, not a few studies about SNP and expression analysis using various tissues of purebred pigs were reported in recently (Davoli et al., 2011; Fontanesi et al., 2011; Huang et al., 2012; Li et al., 2012). Therefore, these results could provide reliable information for studying various target genes related to diseases, meat quality, litter size, reproduction, and development in pigs, and comparative analysis with purebred and crossbred pigs. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.12.052.
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Please cite this article as: Park, S.-J., et al., Selection of appropriate reference genes for RT-qPCR analysis in Berkshire, Duroc, Landrace, and Yorkshire pigs, Gene (2014), http://dx.doi.org/10.1016/j.gene.2014.12.052
