β-actin gene expression is variable among individuals and not suitable for normalizing mRNA levels in Portunus trituberculatus

Zhou, Suming; Zhang, Tao; Shen, Chen; Dong, Qian; Wang, Chunlin; Zhou, Qicun; Jin, Songqing

doi:10.1016/j.fsi.2018.07.021

Cited by 20 publications

(8 citation statements)

References 22 publications

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“…PCR Primers were designed with Primer 3 and Oligo 7.0 (Table S2). Then 18S was used as a housekeeping gene to normalize the mRNA levels of DEGs [31,32]. The qRT-PCR was performed in triplicate by using the Lightcycler 480 II (Roche Applied Science, Penzberg, Germany) with the following reaction conditions: Pre-denaturation at 95 • C for 10 s; amplification 45 cycles of 95 • C for 10 s, 59 • C for 10 s, and 72 • C for 10 s, and using the following program: 95 • C for 10 min; 45 cycles of 95 • C for 10 s, 60 • C for 10 s, and 72 • C for 10 s; 72 • C for 6 min.…”

Section: Real-time Quantitative Reverse Transcription Pcr (Qrt-pcr)mentioning

confidence: 99%

Comparative Transcriptome Analysis of Gonads for the Identification of Sex-Related Genes in Giant Freshwater Prawns (Macrobrachium Rosenbergii) Using RNA Sequencing

Jiang

Yuan

Qingqing

et al. 2019

Genes

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The giant freshwater prawn (Macrobrachium rosenbergii) exhibits sex dimorphism between the male and female individuals. To date, the molecular mechanism governing gonadal development was unclear, and limited data were available on the gonad transcriptome of M. rosenbergii. Here, we conducted comprehensive gonadal transcriptomic analysis of female (ZW), super female (WW), and male (ZZ) M. rosenbergii for gene discovery. A total of 70.33 gigabases (Gb) of sequences were generated. There were 115,338 unigenes assembled with a mean size of 1196 base pair (bp) and N50 of 2195 bp. Alignment against the National Center for Biotechnology Information (NCBI) non-redundant nucleotide/protein sequence database (NR and NT), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, SwissProt database, Protein family (Pfam), Gene ontology (GO), and the eukaryotic orthologous group (KOG) database, 36,282 unigenes were annotated at least in one database. Comparative transcriptome analysis observed that 10,641, 16,903, and 3393 genes were significantly differentially expressed in ZW vs. ZZ, WW vs. ZZ, and WW vs. ZW samples, respectively. Enrichment analysis of differentially expressed genes (DEGs) resulted in 268, 153, and 42 significantly enriched GO terms, respectively, and a total of 56 significantly enriched KEGG pathways. Additionally, 23 putative sex-related genes, including Gtsf1, IR, HSP21, MRPINK, Mrr, and other potentially promising candidate genes were identified. Moreover, 56,241 simple sequence repeats (SSRs) were identified. Our findings provide a valuable archive for further functional analyses of sex-related genes and future discoveries of underlying molecular mechanisms of gonadal development and sex determination. delicious flesh and high value nutrition. Like other crustaceans, M. rosenbergii displays sexual dimorphic growth pattern: The male individuals grow much larger and faster than the females [1,2]. Thus, based on the economic impact, developing the monosex population culture as the efficient approach to boost production has received great attention from researchers in M. rosenbergii. Nevertheless, the premise of monosex culture is to understand the molecular mechanism of sex determination, as well as to unravel sex-related genes.Studies on sex determination have proved that many macruran species exhibit the ZW/ZZ sex determination system [3][4][5]. In M. rosenbergii, sex reversal experiments proposed that the female prawns bear the ZW sex chromosomes, and the male individuals bear the ZZ chromosomes [1,6,7]. Subsequently, isolation of sex-specific markers further deepened the insight of the ZW/ZZ sex determination mode [8,9]. Recently, Ma et al. have confirmed that M. rosenbergii has the ZW/ZZ sex determination system based on the bacterial artificial chromosome (BAC) library, and nine genes were unraveled to be the key sex-linked genes by PacBio sequencing. Amongst, three genes, including zinc knuckle domain (ZKD), reverse transcriptase, and ANCDUO, protein homologs were known genes without characteri...

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Section: Real-time Quantitative Reverse Transcription Pcr (Qrt-pcr)mentioning

confidence: 99%

Comparative Transcriptome Analysis of Gonads for the Identification of Sex-Related Genes in Giant Freshwater Prawns (Macrobrachium Rosenbergii) Using RNA Sequencing

Jiang

Yuan

Qingqing

et al. 2019

Genes

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“…Concentrations of GAPDH , however, may vary among individuals 44 , during pregnancy 45 , according to developmental stage 46 , 47 and during the cell cycle 48 . Other reports also documented such a limitation 49 – 53 . Therefore, the recognition that the expression of Gapdh may exhibit tissue-specific regulationis consistent with our results and emphasizes the need for validation of commercially available control assays.…”

Section: Discussionmentioning

confidence: 82%

Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models

Anauate

Amaral

Ferreira

et al. 2021

Sci Rep

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Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common inherited renal disorder, characterized by renal cyst development leading to end-stage renal disease. Although the appropriate choice of suitable reference is critical for quantitative RNA analysis, no comparison of frequently used “housekeeping” genes is available. Here, we determined the validity of 7 candidate housekeeping genes (Actb, Actg1, B2m, Gapdh, Hprt, Pgam1 and Ppia) in kidney tissues from mouse models orthologous to ADPKD, including a cystic mice (CY) 10–12 weeks old (Pkd1flox/flox:Nestincre/Pkd1flox/−:Nestincre, n = 10) and non-cystic (NC) controls (Pkd1flox/flox/Pkd1flox/-, n = 10), Pkd1-haploinsufficient (HT) mice (Pkd1+/−, n = 6) and wild-type (WT) controls (Pkd1+/+, n = 6) and a severely cystic (SC) mice 15 days old (Pkd1V/V, n = 7) and their controls (CO, n = 5). Gene expression data were analyzed using six distinct statistical softwares. The estimation of the ideal number of genes suggested the use of Ppia alone as sufficient, although not ideal, to analyze groups altogether. Actb, Hprt and Ppia expression profiles were correlated in all samples. Ppia was identified as the most stable housekeeping gene, while Gapdh was the least stable for all kidney samples. Stat3 expression level was consistent with upregulation in SC compared to CO when normalized by Ppia expression. In conclusion, present findings identified Ppia as the best housekeeping gene for CY + NC and SC + CO groups, while Hprt was the best for the HT + WT group.

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“…cDNA was used to measure the relative abundance of mRNA transcript of immune genes, namely prophenoloxidase (ProPO), superoxide dismutase (SOD) and heat shock protein 70 (HSP70) in gills of the shrimps by quantitative real‐time PCR analysis using ABI StepOne Plus thermocycler with 1× Power SYBR Green Master Mix (Applied Biosystems) and gene‐specific primers as reported in previous study (Tomy et al, 2016). The relative transcription value was calibrated with the internal control ef‐1α gene (Leelatanawit et al, 2012; Zhou et al, 2018).…”

Section: Methodsmentioning

confidence: 99%

Effect of dietary C‐Phycocyanin on growth, survival, haematology, immune response, gut microbiome and disease resistance of Pacific white shrimp, Penaeus vannamei

Puthiyedathu

Angel

Sivaramakrishnan

et al. 2022

Aquaculture Research

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C‐Phycocyanin (CPC) was extracted from Arthrospira sp. and isolated from an estuarine ecosystem in southern India. A 45‐day feeding trial was conducted to unravel the effect of dietary CPC in Penaeus vannamei. Seven iso‐nitrogenous and iso‐energetic experimental diets were prepared with different levels of CPC at 0 (CPC0), 50 (CPC50), 100 (CPC100), 200 (CPC200), 400 (CPC400), 800 (CPC800) and 1600 (CPC1600) mg per kg diet. The results revealed that the survival percentages in CPC200, CPC400, CPC800 and CPC1600 were significantly (p < 0.05) higher than in other treatments. Moreover, P. vannamei fed with CPC800 and CPC1600 showed a significantly higher percentage (p < 0.05) of weight gain compared to other treatments. Similarly, compared to other treatments, average body weight, average daily gain, specific growth rate and feed conversion ratio improved significantly (p < 0.05) in CPC800 and CPC1600. Differential gene expression of selected immune genes (ProPO, SOD and HSP70) indicated enhanced immunity in P. vannamei fed with CPC400 and higher CPC doses. The enhanced health of P. vannamei fed with diets containing CPC was further corroborated by the gut Vibrio counts, haematology, gut microbiome and histopathological analyses, and the results showed the addition of CPC at 800 mg per kg of feed enhanced the health of shrimps. The results also give the baseline information about the utilization of CPC in P. vannamei grow‐out feed at an inclusion level of 800 mg per kg of feed to get better growth, survival and health. The results would help in developing functional feeds for shrimps to act against bacterial diseases.

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β-actin gene expression is variable among individuals and not suitable for normalizing mRNA levels in Portunus trituberculatus

Cited by 20 publications

References 22 publications

Comparative Transcriptome Analysis of Gonads for the Identification of Sex-Related Genes in Giant Freshwater Prawns (Macrobrachium Rosenbergii) Using RNA Sequencing

Comparative Transcriptome Analysis of Gonads for the Identification of Sex-Related Genes in Giant Freshwater Prawns (Macrobrachium Rosenbergii) Using RNA Sequencing

Ppia is the most stable housekeeping gene for qRT-PCR normalization in kidneys of three Pkd1-deficient mouse models

Effect of dietary C‐Phycocyanin on growth, survival, haematology, immune response, gut microbiome and disease resistance of Pacific white shrimp, Penaeus vannamei

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