Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in the Half-Smooth Tongue Sole (Cynoglossus semilaevis) at Different Developmental Stages, in Various Tissue Types and on Exposure to Chemicals

Liu, Conghui; Xin, Nian; Zhai, Yi; Jiang, Liming; Zhai, Jieming; Zhang, Quanqi; Qi, Jie

doi:10.1371/journal.pone.0091715

Cited by 35 publications

(38 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Besides, we have analyzed the transcriptome data of tissues and early developmental stages respectively by four criteria. Interestingly, there were 375 and 752 genes that were obtained, suggesting a significant difference in gene expression patterns between tissues and developmental stages, which is similar to some other studies [23][24][25]. After comparing two gene sets obtained from tissues and developmental stages, we obtained a core set of genes (122 genes), most genes are overlapped with the candidate reference genes from 121 gene sets, confirming the stringency and reliability of the methods we applied.…”

Section: Stability Analysis Between Candidates and Commonly Used Refesupporting

confidence: 85%

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

Jin¹,

Song²,

Wang³

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

Background: The quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used technique that relies on the reference gene for gene expression normalization. Selecting a suitable reference gene is a crucial step to obtain an accurate result in qRT-PCR. However, most previous studies of fishes adopted reference genes that were commonly used in mammals without validation. Results: In this study, we utilized 89 transcriptome datasets covering early developmental stages and adult tissues, and carried out transcriptome-wide identification and validation of reference genes in Sebastes schlegelii. Finally, 121 candidate reference genes were identified based on four criteria. Eight candidates (METAP2, BTF3L4, EIF5A1, TCTP, UBC, PAIRB, RAB10, and DLD) and four commonly used reference genes (TUBA, ACTB, GAPDH, RPL17) in mammals were selected for validation via qRT- PCR and four statistical methods (delta-Ct, BestKeeper, geNorm, and NormFinder). The results revealed that the candidate reference genes we recommended are more stable than traditionally used ones. Conclusions: This is the first study to conduct transcriptome-wide identification and validation of reference genes for quantitative RT-PCR in the black rockfish, and lay an important foundation for gene expression analysis in teleost.

show abstract

Section: Stability Analysis Between Candidates and Commonly Used Refesupporting

confidence: 85%

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

Jin¹,

Song²,

Wang³

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…Specific primers MIIB‐qPCR‐DAB‐Fw and MIIB‐qPCR‐DAB‐Rv, MIIB‐qPCR‐DBB‐Fw and MIIB‐qPCR‐DBB‐Rv were used for Cyse‐DAB and Cyse‐DBB , respectively (Table ). Housekeeping gene gapdh was used as the internal reference gene as suggested (Liu et al., ). 2 −∆∆ct was used to calculate the relative expression of class II genes with expression in brain set to 1 in each gene.…”

Section: Methodsmentioning

confidence: 99%

Duplicated major histocompatibility complex class II genes in the tongue sole (Cynoglossus semilaevis)

Jiang

Zhang

et al. 2018

Int J Immunogenetics

View full text Add to dashboard Cite

The major histocompatibility complex (MHC) molecule plays an important role in the vertebrate immune system. However, we have a limited understanding of the MHC genomic structure in teleosts. Using gene cloning and family analysis, we isolate the MHC class II genes in the tongue sole (Cynoglossus semilaevis) and find that both class II A and class II B genes are duplicated (named Cyse-DAA and Cyse-DBA, Cyse-DAB and Cyse-DBB, respectively). The class II A genes consist of four exons with a highly conserved genomic structure, but each gene has unique and defining exon 2 and intron 2 sequences. The class II B genes have a conserved six-exon genomic structure, with intron 3 splitting the β2 encoding region into two exons. Each class II B gene has unique variations in exon 2 and intron 1 sequences. The two class II A genes have similar expression patterns among tissues, with high levels in spleen and gill. Both class II B genes have similar patterns, with high expression in spleen, gill and intestine. The alleles of MHC class II have wide distribution and reliable inheritance in the families analysed. This indicates that the duplicated MHC genes are all classical class II genes. The class II gene duplication with divergent exon and intron sequences, but similar expression patterns in tongue sole provides new insights into MHC evolution.

show abstract

“…low cost and ease of operation, qPCR is consistently used to assess the expression levels of specific genes. As a crucial step requires that the RT-qPCR data is accurately normalized by use of appropriate reference genes, as the use of non-validated reference genes can lead to erroneous results, which are biologically meaningless (28). However, there are currently no universally applicable reference genes.…”

mentioning

confidence: 99%

Selection of suitable reference genes for reverse transcription-quantitative polymerase chain reaction analysis of neuronal cells differentiated from bone mesenchymal stem cells

Yan

Yang

et al. 2015

Molecular Medicine Reports

View full text Add to dashboard Cite

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a technique widely used for the quantification of mRNA transcription, It is well recognized that the reference genes used in RT-qPCR require appropriate validation to ensure that gene expression is unaffected by experimental conditions. The differentiation of bone mesenchymal stem cells (BMSCs) into neurons is important in the treatment of nerve injury. In gene expression analysis of the differentiation of BMSCs into neuronal cells by, the commonly used reference genes for RNA analysis are often selected without any preliminary evaluation of their suitability. The present study aimed to evaluate the mRNA expression levels of 11 putative reference genes, including ACTB, ARBP, B2M, CYCA, GAPDH, GUSB, HPRT, PPIA, RPL13A, TBP and PGK1, in order to select the most suitable reference genes for RT-qPCR of the differentiation of neuronal cells by BMSCs. The mRNA expression levels of the 11 putative reference genes were examined using RT-qPCR in rat BMSCs differentiated into neuronal cells. Normal BMSCs and three types of rat BMSCs, which were chemically induced to differentiate into neurons using neurotrophic cytokines and co-culture with retinal cells. The geNorm, NormFinder and BestKeeper software programs were used to select the most suitable reference genes. The results of the analyses using the three software programs demonstrated that RPL13A was the most stable among all the groups, while ACTB was the least stable. The combination of CYCA and PPIA reference genes contributed the most to increasing stability. The suitability of selected reference genes requires previous pre-selection in every investigation. Based on the three software programs, RPL13A, and the combination of CYCA and PPIA were identified as the most suitable reference genes for RT-qPCR in neuronal cells differentiated from BMSCs.

show abstract

Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in the Half-Smooth Tongue Sole (Cynoglossus semilaevis) at Different Developmental Stages, in Various Tissue Types and on Exposure to Chemicals

Cited by 35 publications

References 46 publications

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

Duplicated major histocompatibility complex class II genes in the tongue sole (Cynoglossus semilaevis)

Selection of suitable reference genes for reverse transcription-quantitative polymerase chain reaction analysis of neuronal cells differentiated from bone mesenchymal stem cells

Contact Info

Product

Resources

About