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

Gromboni, Juliana Gracielle Gonzaga; Oliveira, Hellenn Cardoso; Marques, Daniele Botelho Diniz; García, Adriana; Filho, Ronaldo Vasconcelos Farias; Gromboni, Caio Fernando; Souza, Teillor Machado; Wenceslau, Amauri Arias

doi:10.1371/journal.pone.0228314

Cited by 8 publications

(10 citation statements)

References 40 publications

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“…In addition, expression levels of Hprt1, Actb, and 18 s rRNA were significantly altered following acute HS on porcine iSCs. Although HS has been shown to influence reference gene stability in different chicken tissues (Cedraz de Oliveira et al, 2017; Gromboni et al, 2020), and in blood of Bama miniature pigs (Ju et al, 2011), suitable reference genes of porcine iSCs under acute HS have not been reported yet.…”

Section: Introductionmentioning

confidence: 99%

Identification of internal reference genes for porcine immature Sertoli cells under heat stress

Wang

Fang

et al. 2022

Reprod Domestic Animals

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Identification of stably expressed gene(s) as internal reference(s) for different experimental conditions is key to the accurate normalization and quantification of target transcripts. Previously, our RNA‐seq study showed that Hprt1, Actb, and 18S rRNA abundances were all significantly altered in porcine immature Sertoli cells (iSCs) during acute heat stress (HS). In the current study, we aimed to identify stable reference gene(s) to study the gene expression dynamics of quick and delayed responses after acute HS treatment of porcine iSCs. A total of six genes previously used in pig testis or Sertoli cells (Hprt1, Top2b, Actb, Rpl32, Gapdh, and 18S rRNA) were chosen to perform RT‐qPCR for the control (before acute HS), HS0.5 (acute HS at 43°C for 0.5 h), and HS0.5‐R36 (36 h recovery following acute HS) groups. The stability of candidate reference genes was examined by the GeNorm, NormFinder, BestKeeper and Comparative ΔCt methods, and RefFinder to obtain the final rank. Rpl32 and Actb were the two most stable internal reference genes as found by all methods, whereas Hprt1 and 18S rRNA were the two most unstable as ranked by RefFinder. Moreover, expression of six target mRNAs (Ccn1, Ccnb1, Eif4g1, Hdac6, Plk2, and Ptma) was normalized using Rpl32, Actb, or the combination of Rpl32 and Actb, respectively. Therefore, our findings that the most suitable internal references are Rpl32 and Actb provide useful information for further functional investigation on genes regulating the acute HS of porcine iSCs.

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Section: Introductionmentioning

confidence: 99%

Identification of internal reference genes for porcine immature Sertoli cells under heat stress

Wang

Fang

et al. 2022

Reprod Domestic Animals

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“…Previous studies have reported relatively high variability for the expression of WU-HKGs in a varied range of tissues and organs in livestock animals ( Gromboni et al, 2020 ; Lozano-Villegas et al, 2021 ) as well as in humans ( Mahoney et al, 2004 ; Caracausi et al, 2017 ; Cadenas et al, 2022 ), mice ( Fu et al, 2020 ; Muñoz et al, 2021 ), and insects ( Shi et al, 2016 ), among others.…”

Section: Resultsmentioning

confidence: 99%

“…On that account, only 33 WU-HKGs were analyzed. Gromboni et al (2020) assessed the suitability of eight HKGs for heart. Their studied WU-HKGs were present in neither the top 10 genes nor the list of 23 V-HKGs of heart tissue in the current study.…”

Section: Resultsmentioning

confidence: 99%

“…To our knowledge, the present work is the first comprehensive study that investigated all REGs for 16 most important chicken tissues. Most of the previous studies have compared only a handful of HKGs in which the used genes were in common ( Zinzow-Kramer et al, 2014 ; Bagés et al, 2015 ; Mitra et al, 2016 ; Khan et al, 2017 ; Hassanpour et al, 2018 ; Zhang et al, 2018 ; Gromboni et al, 2020 ). The employed methodologies of the mentioned studies were NormFinder, GeNorm, BestKeeper, RefFinder, and delta CT ( Mitra et al, 2016 ).…”

Section: Resultsmentioning

confidence: 99%

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Investigation of chicken housekeeping genes using next-generation sequencing data

et al. 2022

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Accurate normalization of the gene expression assays, using housekeeping genes (HKGs), is critically necessary. To do so, selection of a proper set of HKGs for a specific experiment is of great importance. Despite many studies, there is no consensus about the suitable set of HKGs for implementing in the quantitative real-time PCR analyses of chicken tissues. A limited number of HKGs have been widely used. However, wide utilization of a little number of HKGs for all tissues is challenging. The emergence of high-throughput gene expression RNA-seq data has enabled the simultaneous comparison of the stability of multiple HKGs. Therefore, employing the average coefficient of variations of at least three datasets per tissue, we sorted all reliably expressed genes (REGs; with FPKM ≥ 1 in at least one sample) and introduced the top 10 most suitable and stable reference genes for each of the 16 chicken tissues. We evaluated the consistency of the results of five tissues using the same methodology on other datasets. Furthermore, we assessed 96 previously widely used HKGs (WU-HKGs) in order to challenge the accuracy of the previous studies. The New Tuxedo software suite was used for the main analyses. The results revealed novel, different sets of reference genes for each of the tissues with 17 common genes among the top 10 genes lists of 16 tissues. The results did disprove the suitability of WU-HKGs such as Actb, Ldha, Scd, B2m, and Hprt1 for any of the tissues examined. On the contrary, a total of 6, 13, 14, 23, and 32 validated housekeeping genes (V-HKGs) were discovered as the most stable and suitable reference genes for muscle, spleen, liver, heart, and kidney tissues, respectively. Although we identified a few new HKGs usable for multiple tissues, the selection of suitable HKGs is required to be tissue specific. The newly introduced reference genes from the present study, despite lacking experimental validation, will be able to contribute to the more accurate normalization for future expression analysis of chicken genes.

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“…The stability rank order in the tissue groups was different from that observed in the acaricide treatment groups, with α‐tubulin and RpL5 being the most stable and Actin and eEF2 being the least stable. α‐tubulin encodes a major component of the cytoskeleton and has been widely accepted as a reference gene across various species (P. Liang et al, 2014; Rentoft et al, 2010; Sun et al, 2010) along with RpL5 , which encodes a ribosomal subunit protein (Gromboni et al, 2020; Hul et al, 2020). Both α‐tubulin and RpL5 showed good performance in all the cross‐tissue comparisons, except for the cuticle‐gut/fat body comparison, indicating that the application of both genes as reference genes for cross‐tissue comparison is reliable.…”

Section: Discussionmentioning

confidence: 99%

Selection of stable reference genes for quantitative real‐time PCR in the Varroa mite, Varroa destructor

Lee

Kim

et al. 2022

Arch Insect Biochem Physiol

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To investigate the acaricide toxicity and resistance mechanisms in the Varroa mite, it is essential to understand the genetic responses of Varroa mites to acaricides, which are usually evaluated by transcriptional profiling based on quantitative real‐time polymerase chain reaction (qPCR). In this study, to select reference genes showing consistent expression patterns regardless of the acaricide treatment or the type of tissue, Varroa mites treated with each of the three representative acaricides (coumaphos, fluvalinate, and amitraz) were processed for transcriptomic analysis, from which eight genes (NADH dehydrogenase [NADHD], glyceraldehyde‐3‐phosphate dehydrogenase [GAPDH], eukaryotic translation elongation factor 1 α 1 [eEF1A1], eukaryotic translation elongation factor 2 [eEF2], ribosomal protein L5 [RpL5], Actin, tubulin α‐1D chain [α‐tubulin], and Rab1) were selected as candidates. The transcription profiles of these genes, depending on the treatment of the three acaricides or across different tissues (cuticle, legs, gut/fat bodies, and synganglion), were analyzed using qPCR with four validation programs, BestKeeper, geNorm, NormFinder, and RefFinder. Following acaricide treatment, eEF1A1 and NADHD showed the least variation in their expression levels, whereas the expression levels of α‐tubulin and RpL5 were the most stable across different tissue groups. Rab1/GAPDH and Actin/eEF2 showed the least stable expression patterns following acaricide treatments and across different tissues, respectively, requiring precautions for use. When vitellogenin gene expression was analyzed by different reference genes, its expression profiles varied significantly depending on the reference genes, highlighting the importance of proper reference gene use. Thus, it is recommended using eEF1A1 and NADHD as reference genes for the comparison of the effects of acaricide on the whole body, whereas α‐tubulin and RpL5 are recommended for investigating the tissue‐specific expression profiles of target genes.

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

Abstract: Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here:

Cited by 8 publications

References 40 publications

Identification of internal reference genes for porcine immature Sertoli cells under heat stress

Identification of internal reference genes for porcine immature Sertoli cells under heat stress

Investigation of chicken housekeeping genes using next-generation sequencing data

Selection of stable reference genes for quantitative real‐time PCR in the Varroa mite, Varroa destructor

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