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On-line version ISSN 1678-4685
Genet. Mol. Biol. vol.30 no.3 São Paulo 2007
Lilian Giotto ZarosI; Patrízia Ana BricarelloII; Alessandro Francisco Talamini AmaranteII; Luiz Lehmann CoutinhoI
ILaboratório de Biotecnologia Animal, Departamento de Zootecnia, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, Brazil
IIDepartamento de Parasitologia, Universidade Estadual Paulista, Botucatu, SP, Brazil
T cells produce cytokines that affect host response to infection. This paper reports real-time RT-PCR conditions and validation steps for accurate quantification of Bos indicus cytokines, interleukin (IL)-2, IL-4, IL-8, IL12p-35, IL-13, tumoral necrosis factor (TNF)-a, interferon (IFN)-g, monocyte chemoattractant proteins (MCP)-1 and MCP-2, and the glycoprotein mucin (MUC)-1 in two groups of Nelore cattle, one resistant and the other susceptible to gastrointestinal nematode infections. RPL-19 was shown to be an ideal internal control gene, since its expression was constant across treatments and presented lower variation when compared to the GAPDH gene. The optimized conditions established in the present study can be used to determine the immune response of cattle under different experimental conditions, such as viral, bacterial and parasite infections.
Key words: cytokine gene expression, real-time RT-PCR, cattle, gastrointestinal nematodes, bovine cytokines.
The vertebrate immune system has evolved to defend the host against several kinds of pathogens, including gastrointestinal nematodes (Finkelman et al., 2004). The T cells, conductors of this response, can be divided into TH1 and TH2, and their development is influenced by several cytokines, among them interleukin (IL)-4 and interferon (IFN)-g. IL-4 promotes TH2 cell expansion and limits TH1 cell proliferation. In contrast, IFN-g induces TH1 and inhibits TH2 response (Liew, 2002). TH1 response has been associated with host susceptibility to nematode infection, while TH2 response permits the host to build up a protective reaction, facilitating the expulsion of parasites, and has been observed in resistant animals (Finkelman et al., 2004). Consequently, the study of cytokine gene expression is important for understanding the immune response.
Real-time RT - PCR is a highly sensitive method that allows quantification of rare transcripts and small changes in gene expression from a limited amount of sample (Leutenegger et al., 1999; Konnai et al., 2003). However, reliable gene expression quantification using the RT-PCR assay requires specific primers for the target gene and correction for the efficiency of DNA amplification. A control gene that is not affected by the experimental treatment must be used to correct for variations due to differences in RNA quantity, as well as efficiency of reverse transcription and cDNA amplification (Giulietti et al., 2001).
Real-time RT-PCR has been used to measure cytokine gene expression in mammals; however, there are few reports in cattle, and these are limited to Bos taurus (Konnai et al., 2003; Gómez-Muñoz et al., 2004; Claerebout et al., 2005). In the present study, we determined optimal real-time RT-PCR conditions to quantify Bos indicus cytokines, interleukin (IL)-2, IL-4, IL-8, IL12p-35, IL-13, tumoral necrosis factor (TNF)-a, interferon (IFN)-g, monocyte chemoattractant protein (MCP)-1 and MCP-2, and the glycoprotein mucin (MUC)-1, in two groups of Nelore cattle, one resistant and the other susceptible to gastrointestinal nematode infections.
Animals and sample preparation
A group of 100 Bos indicus bulls (Nelore breed) aged 11 to 12 months were kept together on pasture without anthelmintic treatment for 6 months on a farm located in Londrina, State of Paraná, in southern Brazil (longitude 23°18'37" S, latitude 51°09'46'' O). They were kept on 79 hectares (divided in 4 paddocks) of Panicum maximum pastures, naturally contaminated by nematode larvae. Feces samples were taken 7 times at a two-weeks interval from all animals, to determine the fecal egg counts (FEC). The mean FEC of each animal was calculated, and the 10 animals with the lowest mean FEC (31.6 ± 43.1; called resistant - R) and 10 with the highest mean FEC (674.6 ± 505.7; called susceptible - S) were selected and slaughtered. Tissue samples from the abomasum, small intestine and abomasum lymph nodules of these animals were collected immediately after slaughter. Total RNA was isolated using Trizol Reagent (Invitrogen Co Carlsbad, CA, USA) according to the manufacturer's protocol. The RNA concentration was assessed by spectrophotometry at 260 nm, and RNA quality was assessed by the OD260nm:OD280nm ratio and electrophoresis on 1% agarose gel stained with ethidium bromide.
Primers were designed using Primer3 primer design software (Rozen and Skaletzky, 2000) and cattle-specific GenBank sequences for IL-2 (AF535144), IL-4 (M77120), IL-8 (AF061521), IL-12p35 (AJ271034), IL-13 (NM174089), TNF-a (AF011926), IFN-g (Z54144), MCP-1 (L32659), MCP-2 (S67956), and MUC-1 (AF399757). The internal control genes tested were ribosomal protein (RP) L-19 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primers were analyzed using Oligo Analysis & Plotting Tools software from Operon, in order to avoid secondary structures such as hairpins and loops. Primers were designed on different exons, to differentiate cDNA and genomic DNA amplification. Primer sequences and amplicon lengths are shown in Table 1.
Confirmation of primer specificity
Amplification and melting analysis were performed on a LightCycler (Roche Diagnostics, Mannheim, Germany). Specificity of the RT-PCR products was evaluated by agarose gel electrophoresis, melting curve analysis and direct sequence of amplified products. Melting curve analysis was performed from 70 °C to 95 °C at 0.1 °C/s for all genes studied. Melting temperature (TM) was defined as the peak of the curve, and if the highest point was a plateau, then the midpoint was identified as TM. Sequencing was done with the DYEnamic ET DyeTerminator Sequencing Kit (Amersham Bioscience, Piscataway, NJ, USA) and the ABI 3100 Automated DNA Sequencer (Applied Biosystems).
Serial cDNA dilution curves were produced to calculate the amplification efficiency for all genes. A graph of threshold cycle (Ct) versus log10 relative copy number of the sample from a dilution series was produced. The slope of the curve was used to determine the amplification efficiency (Pfaffl, 2001): Efficiency = 10 (-1/slope).
cDNA was reverse-transcribed from 5.0 µg total RNA using oligo(dT) primer and Superscript II (Invitrogen Co Carlsbad, CA, USA), following the manufacturer's instructions. Real-time PCR was performed using LightCycler (Roche Diagnostics, Mannheim, Germany) and SYBR Green I (Roche Diagnostics, Mannheim, Germany), according to the manufacturer's instructions. The PCR conditions were: 7.5 ng of cDNA, MgCl2 at optimal concentration for each gene, 2-2.5 pmol of each primer, 4 µL of SYBR Green I, 0.3 µL of Taq Platinum DNA polymerase (Invitrogen, 5U/mL), 0.4 µL of dNTP (10 mM), and 0.25 µL BSA (20 mg/mL), in a final volume of 20 µL. Reactions were incubated in a LightCycler, following specific conditions for each gene (Table 1). Initial denaturation and denaturation step were the same for all primers, i.e., 95 °C for 5 min and 95 °C for 10 s, respectively. The fluorescence acquisition temperature was 72 °C for all genes. The DRn value (fluorescence variation during the PCR reaction) for each gene was 0.1, which was as low as possible without including any background points.
The RT-PCR conditions established resulted in expected amplicon size and single-band amplification for all genes, indicating that the primers and conditions used were specific for the target genes (Figure 1). Amplification specificity was further validated by melting curve analysis, generated at the end of each PCR reaction. All genes presented a single peak in the melting curve, which indicates absence of primer-dimer formation during the reaction and specificity of the amplification. The melting temperature for all genes is presented in Table 1. Melting curve analysis is a fast method to check for RT-PCR specificity; however, in some cases, RT-PCR products of different lengths have the same melting temperature and therefore are indistinguishable by melting curve analysis. To guarantee specificity, the amplified products were sequenced and gene identity was confirmed (data not shown).
The efficiency, sensitivity and linearity of RT-PCR reactions were examined using 10-fold serial dilutions. The relationship between threshold cycle (Ct) and the log copy number of cDNA for all genes was linear with an R2 ranging from 0.96 to 0.99, indicating that Ct values changed proportionally to the serial dilution of the samples. Efficiency values ranged from 1.86 to 2.18, indicating efficient amplification near the theoretical optimum level of 2 (Wilkening and Bader, 2004).
A reliable quantitative RT-PCR method requires correction for experimental variations. In the present study, two internal control genes were tested in the two groups of animals, resistant and susceptible to gastrointestinal nematode infections, and in each one of the three tissues collected (Table 2). Ct values in the different tissues and groups ranged from 15.8 to 21.1 for RPL-19 and from 27.5 to 35.4 for GAPDH. The lower Ct value for RPL-19 indicates that this gene reaches the detection threshold with less amplification cycles than GAPDH, indicating that it is more abundant in the abomasum, abomasum lymph nodules and small intestine. The coefficient of variation ranged from 5.6 to 8.9 for RPL-19 and from 7.5 to 18.5 for GAPDH across all tissues and groups. The expression levels of RPL-19 and GAPDH were not different in resistant and susceptible animals (p > 0.1), however they were differentially expressed among tissues (p < 0.01). These results are in agreement with those of Al-Bader and Al-Sarraf (2005) showing that GAPDH, albumins, actins, tubulins, cyclophilins, 18S rRNA and 28S RNA, used extensively as control genes, may vary under different experimental conditions in different tissues. Although GAPDH is widely used as an internal control gene (Giulietti et al., 2001; Bustin, 2002; Bustin et al., 2005), several reports emphasize problems associated with its use (Ke et al., 2000; Suzuki et al., 2000). Al-Bader and Al-Sarraf (2005) used RT-PCR to measure mRNA levels of several genes, in order to identify those most suitable to normalize expression. These authors concluded that GAPDH was not a suitable control gene, while 18S, RPL-19, BGLU, CY and HH4 were reasonably stable genes that were appropriate for normalization when quantifying mRNA levels. Ontsuka et al. (2004) tested the 18S, RPL-19 and b-actin genes in intestinal fractions and concluded that they were stable.
Several methods to quantify cytokines have been developed and RT-PCR is one of the most important techniques for analyzing immune response in bovines and other ruminants. Konnai et al. (2003) used this method to quantify cytokine profiles (IL-1, IL-2, IL-4, IL-6, IL-10, IL-12p40, and IFN-g) in peripheral blood mononuclear cells from bovine and ovine leukemia virus (BLV)-infected animals and concluded that this methodology is a useful tool in the quantitative analysis of various genes in cattle and sheep. Gómez-Muñoz et al. (2004) analyzed the immune response during bovine ostertagiosis and observed decreased IL-1 IL-2, IL-4, IL-13, TNF-a and IFN-g levels and increased IL-10 and TGF-b levels after challenge, concluding that RT-PCR is a rapid and accurate method to classify Ostertagia ostertagi as a suppressor of in vivo immune response in cattle. Claerebout et al. (2005) studied the abomasal (mucosal and lymph nodules) cytokine response (IL-2, IL-4, IL-5, Il-6, IL-10, IL-12p40, IL-13, IL-15, and IFN-g) in helminthes-naive calves and calves vaccinated with protective antigen from Ostertagia ostertagi after challenge with infective third-stage larvae and concluded that RT-PCR is ideal to obtain reliable data under different experimental conditions, i.e, naive and vaccinated calves.
Real-time RT - PCR has significantly extended the use of RT-PCR assays. The primers and conditions optimized in the present study were sensitive, fast and reliable in the detection and quantification of cytokine mRNAs, and the RPL-19 gene was demonstrated to be an ideal internal control gene in bovine experiments. Such real-time PCR assays can be used in Bos indicus cattle to investigate the bovine immune response in a variety of situations, such as viral, bacterial and parasite infections.
This work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP). (Lilian Giotto Zaros and Patrízia Ana Bricarello received financial support from FAPESP.) Luiz Lehmann Coutinho is a recipient of a research productivity scholarship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
Al-Bader MD and Al-Sarraf HA (2005) Housekeeping gene expression during fetal brain development in the rat - Validation by semi-quantitative RT-PCR. Dev Brain Res 156:38-45. [ Links ]
Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR); trends and problems. J Mol Endocrinol 29:23-39. [ Links ]
Bustin SA, Benes A, Nolan T and Pfaffl MW (2005) Quantitative real-time RT-PCR - A perspective. J Mol Endocrinol 34:597-601. [ Links ]
Claerebout E, Vercauteren I, Geldhof P, Olbrechts A, Zarlenga DS, Goddeeris S and Vercruysse BM (2005) Cytokine response in immunized and non-immunized calves after Ostertagia ostertagi infection. Parasite Immunol 27:325-331. [ Links ]
Finkelman FD, Shea-Donohue T, Morris SC, Gildea L, Strait R, Madden KB, Schopf L and Urban Jr JF (2004) Interleukin-4-and interleukin-13-mediated host protection against intestinal nematode parasites. Immunol Rev 201:139-155. [ Links ]
Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R and Mathieu C (2001) An overview of real time quantitative PCR: Applications to quantify cytokine gene expression. Methods 25:386-401. [ Links ]
Gómez-Muñoz MT, Canals-Caballero A, Almeria S, Pasquali P, Zarlenga DS and Gasbarre LC (2004) Inhibition of bovine T lymphocyte response by extracts of the stomach worm Ostertagia ostertagi. Vet Parasitol 120:199-214. [ Links ]
Ke LD, Chen Z and Yung WKA (2000) A reliability test of standard-based quantitative PCR: Exogenous vs. endogenous standards. Mol Cell Probes 14:127-135. [ Links ]
Konnai S, Usui T, Ohashi K and Onuma M (2003) The rapid quantitative analysis of bovine cytokine genes by real-time RT-PCR. Vet Microbiol 94:283-294. [ Links ]
Leutenegger CM, Mislin CN, Sigrist B, Ehrengruber MU, Hofmann-Lehmann R and Lutz H (1999) Quantitative real-time PCR for measurement of feline cytokine mRNA. Vet Immunol Immunopathol 71:291-305. [ Links ]
Liew FY (2002) Th1 and Th2 cells: A historical perspective. Nat Rev 2:55-59. [ Links ]
Ontsuka EC, Korczak B, Hammon HM and Blum JW (2004) Real-time PCR quantification of bovine lactase mRNA: Localization in the gastrointestinal tract of milk-fed calves. J Dairy Sci 87:4230-4237. [ Links ]
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:2002-2007. [ Links ]
Rozen S and Skaletzky HJ (2000) Primer 3 on the WWW for General Users and for Biologist Programmers. Humana Press, Totowa, 568 pp. [ Links ]
Suzuki T, Higgens PJ and Crawford DR (2000) Control selection for RNA quantification. Biotechniques 29:332-337. [ Links ]
Wilkening S and Bader A (2004) Quantitative real-time polymerase chain reaction: Methodical analysis and mathematical model. J Biomol Tech 15:107-111. [ Links ]
Operon Biotechnologies GmbH: https://www.operon.com/ oligos/toolkit.php.
Send correspondence to:
Luiz Lehmann Coutinho
Laboratório de Biotecnologia Animal
Departamento de Zootecnia
Escola Superior de Agricultura Luiz de Queiroz
Universidade de São Paulo
Avenida Pádua Dias 11
13480-900 Piracicaba, SP, Brazil
Received: June 27, 2006; Accepted: January 23, 2007.
Associate Editor: Pedro Franklin Barbosa