Influence of heat stress on reference genes stability in heart and liver of two chickens genotypes.

INTRODUCTION:Real-time polymerase chain reaction (RT-qPCR) is an important tool for analyzing gene expression. However, before analyzing the expression of target genes, it is crucial to normalize the reference genes, in order to find the most stable gene to be used as an endogenous control. A gene that remains stable in all samples under different treatments is considered a suitable normalizer. In this sense, we aimed to identify stable reference genes for normalization of target genes in the heart and liver tissues from two genetically divergent groups of chickens (Cobb 500® commercial line and Peloco backyard chickens) under comfort and acute heat stress environmental conditions. Eight reference genes (ACTB, HPRT1, RPL5, EEF1, MRPS27, MRPS30, TFRC and LDHA) were analyzed for expression stability. The samples were obtained from 24 chickens, 12 from the backyard Peloco and 12 from the Cobb 500® line, exposed to two environmental conditions (comfort and heat stress). Comfort temperature was 23°C and heat stress temperature was 39.5°C for one hour. Subsequently, the animals were euthanized, and heart and liver tissue fragments were collected for RNA extraction and amplification. To determine the stability rate of gene expression, three different statistical algorithms were applied: BestKeeper, geNorm and NormFinder, and to obtain an aggregated stability list, the RankAgregg package of R software was used. RESULTS:The most stable genes using BestKeeper tool, including the two factors (genetic group and environmental condition), were LDHA, RPL5 and MRPS27 for heart tissue, and TFRC, RPL5 and EEF1 for liver tissue. Applying geNorm algorithm, the best reference genes were RPL5, EEF1 and MRPS30 for heart tissue and LDHA, EEF1 and RPL5 for liver. Using the NormFinder algorithm, the best normalizer genes were EEF1, RPL5 and LDHA in heart, and EEF1, RPL5 and ACTB in liver tissue. In the overall ranking obtained by RankAggreg package, considering the three algorithms, the RPL5, EEF1 and LDHA genes were the most stable for heart tissue, whereas RPL5, EEF1 and ACTB were the most stable for liver tissue. CONCLUSION:According to the RankAggreg tool classification based on the three different algorithms (BestKeeper, geNorm and NormFinder), the most stable genes were RPL5, EEF1 and LDHA for heart tissue and RPL5, EEF1 and ACTB for liver tissue of chickens subjected to comfort and acute heat stress environmental conditions. However, the best reference genes may vary depending on the experimental conditions of each study, such as different breeds, environmental stressors, and tissues analyzed. Therefore, the need to perform priori studies to assay the best reference genes at the outset of each study is emphasized

Influence of heat stress, sex and genetic groups on reference genes stability in muscle tissue of chicken

<div><p>Quantitative RT-PCR is an important technique for assessing gene expression. However, a proper normalization of reference genes prior to expression analyses of target genes is necessary. The best normalizer is that gene which remains stable in all samples from different treatments. The aim of this study was to identify stable reference genes for normalization of target genes in muscle tissue from three genetically divergent chickens groups (Peloco, Cobb 500^® and Caneluda) under environmental (heat stress and comfort) and sex influence. Expressions of ten reference genes were tested for stability in breast muscular tissue (<i>Pectoralis major</i> muscle). Samples were obtained from 36 males and females of two backyard breeds (Caneluda and Peloco) and one commercial line (Cobb 500^®) under two environments. The heat stress and comfort temperature were 39 and 23°C, respectively. Animals were housed in the Animal Science Department at <i>Universidade Estadual do Sudoeste da Bahia</i>. We analyzed the expression data by four statistical tools (SLqPCR, NormFinder, Bestkeeper and Comparative CT). According to these tools, genes stability varied according to sex, genetic group and environment, however, some genes remained stable in all analyzes. There was no difference between the most stable genes for sex effect, being <i>MRPS27</i> more stable for both males and females. In general, <i>MRPS27</i> was the most stable gene. Within the three genetic groups, the most stable genes were <i>RPL5</i>, <i>HMBS</i> and <i>EEF1</i> to Cobb 500^®, Peloco and Caneluda, respectively. Within the environment, the most stable gene under comfort and heat stress conditions was <i>HMBS</i> and <i>MRPS27</i>, respectively. BestKeeper and Comparative Ct were less correlated (28%) and SLqPCR and NormFinder were the most correlated (98%). <i>MRPS27</i>, <i>RPL5</i> and <i>MRPS30</i> genes were considered stable according the overall ranking and can be used as normalizer of relative expression of target genes in muscle tissue of chickens under heat stress.</p></div

Description of <i>Gallus gallus</i> reference genes, their specific primers used in RT-qPCR analysis and parameters derived from RT-qPCR analysis.

<p>All primers were designed by NASCIMENTO et al., (2015).</p

Ranking with stability values for each treatment (genetic group, sex, and environment) in chicken obtained from BestKeeper tool.

<p>Values into the parenthesis refer to ranking by Bestkeeper to each treatment. Values on the last column refer to the ranking position by RankAggreg package among all the treatments.</p

Overall ranking of reference genes in chickens obtained by different tools (SLqPCR, NormFinder, Bestkeeper and Comparative Ct) and ranked by RankAggreg package according to each treatment (genetic groups, environment and sex).

<p>Overall ranking of reference genes in chickens obtained by different tools (SLqPCR, NormFinder, Bestkeeper and Comparative Ct) and ranked by RankAggreg package according to each treatment (genetic groups, environment and sex).</p

Initial feed used in the production of chicks up to 30 days of age (ROSTAGNO, GOMES, 2011).

<p>Initial feed used in the production of chicks up to 30 days of age (ROSTAGNO, GOMES, 2011).</p

ANOVA analysis of Ct values of genetic group, sex and environment effects among previous chosen genes.

<p>ANOVA analysis of Ct values of genetic group, sex and environment effects among previous chosen genes.</p

Descriptive statistics of expression levels of reference genes for chickens obtained by BestKeeper (n = 36).

<p>Descriptive statistics of expression levels of reference genes for chickens obtained by BestKeeper (n = 36).</p

Parameters of reference genes primers specific for chickens obtained from efficiency curve analysis of RT-qPCR.

<p>Parameters of reference genes primers specific for chickens obtained from efficiency curve analysis of RT-qPCR.</p