Transcriptome‐Based Identification of the Optimal Reference CHO Genes for Normalisation of qPCR Data

Brown, Adam; Gibson, Suzanne J.; Hatton, Diane; James, David C.

doi:10.1002/biot.201700259

Cited by 32 publications

(34 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The metabolic changes associated with the cell size increase were studied in detail using flux balance analysis; however, the cause and molecular mechanisms for the cell size increase remained unclear. Transcriptome analysis has been used as a powerful tool to better understand the physiology of CHO cell lines . The aim of the present study is to obtain more insights into the cause and the molecular mechanisms underlying the CHO cell size increase using transcriptome analysis.…”

Section: Introductionmentioning

confidence: 99%

Transcriptome Analysis of CHO Cell Size Increase During a Fed-Batch Process

et al. 2018

View full text Add to dashboard Cite

In a Chinese Hamster Ovary (CHO) cell fed-batch process, arrest of cell proliferation and an almost threefold increase in cell size occurred, which is associated with an increase in cell-specific productivity. In this study, transcriptome analysis is performed to identify the molecular mechanisms associated with this. Cell cycle analysis reveals that the cells are arrested mainly in the G /G phase. The cell cycle arrest is associated with significant up-regulation of cyclin-dependent kinases inhibitors (CDKNs) and down-regulation of cyclin-dependent kinases (CDKs) and cyclins. During the cell size increase phase, the gene expression of the upstream pathways of mechanistic target of rapamycin (mTOR), which is related to the extracellular growth factor, cytokine, and amino acid conditions, shows a strongly synchronized pattern to promote the mTOR activity. The downstream genes of mTOR also show a synchronized pattern to stimulate protein translation and lipid synthesis. The results demonstrate that cell cycle inhibition and stimulated mTOR activity at the transcriptome level are related to CHO cell size increase. The cell size increase is related to the extracellular nutrient conditions through a number of cascade pathways, indicating that by rational design of media and feeds, CHO cell size can be manipulated during culture processes, which may further improve cell growth and specific productivity.

show abstract

Section: Introductionmentioning

confidence: 99%

Transcriptome Analysis of CHO Cell Size Increase During a Fed-Batch Process

et al. 2018

View full text Add to dashboard Cite

show abstract

“…(1) FPKM. Potential HKGs were relatively highly expressed genes [8]. In this study, 5623 genes had FPKM values ≥ 10 (35.5% of 15853 genes, the green area in Figure 1A).…”

Section: Selection Of Novel Candidate Hkgs Based On Rna-seq Datamentioning

confidence: 63%

Transcriptome-based Selection and Validation of Optimal House-keeping Genes for Skin Research in Goats (Capra hircus)

Zhang

Deng

et al. 2020

Preprint

View full text Add to dashboard Cite

Abstract Background : In quantitative real-time polymerase chain reaction (qRT-PCR) experiments, accurate and reliable target gene expression results are dependent on optimal amplification of house-keeping genes (HKGs). RNA-seq technology offers a novel approach to detect new HKGs with improved stability. Goat ( Capra hircus ) is an economically important livestock species and plays an indispensable role in the world animal fiber and meat industry. Unfortunately, uniform and reliable HKGs for skin research have not been identified in goat. Therefore, this study seeks to identify a set of stable HKGs for the skin tissue of C. hircus using high-throughput sequencing technology. Results: Based on the transcriptome dataset of 39 goat skin tissue samples, 8 genes ( SRP68 , NCBP3 , RRAGA , EIF4H , CTBP2 , PTPRA , CNBP , and EEF2 ) with relatively stable expression levels were identified and selected as new candidate HKGs. Commonly used HKGs including SDHA and YWHAZ from a previous study, and 2 conventional genes ( ACTB and GAPDH ) were also examined. Four different experimental variables: (1) different development stages, (2) hair follicle cycle stages, (3) breeds, and (4) sampling sites were used for determination and validation. Four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method) and a comprehensive algorithm (ComprFinder, developed in-house) were used to assess the stability of each HKG. It was shown that NCBP3+SDHA+PTPRA were more stably expressed than previously used genes in all conditions analysis, and that this combination was effective at normalizing target gene expression. Moreover, a new algorithm for comprehensive analysis, ComprFinder, was developed and released. Conclusion: This study presents the first list of candidate HKGs for C. hircus skin tissues based on an RNA-seq dataset. We propose that the NCBP3+SDHA+PTPRA combination could be regarded as a triplet set of HKGs in skin molecular biology experiments in C. hircus and other closely related species. In addition, we also encourage researchers who perform candidate HKG evaluations and who require comprehensive analysis to adopt our new algorithm, ComprFinder.

show abstract

“…(1) FPKM. Potential HKGs were relatively highly expressed genes (expression levels ≥ the 80th percentile or FPKM ≥ 10) [8]. In this study, including 5623 genes with FPKM values > 10 (35.5% of 15853 genes, the green area in Fig.…”

Section: Selection Of Novel Candidate Hkgs Based On Rna-seq Datamentioning

confidence: 86%

“…There are two commonly held misconceptions with regard to the selection of HKGs: (I) HKGs are selected based on experience without verification or without reviewing HKG research papers, and (II) using a single HKG with poor stability. In recent years, it has been reported with increasing frequency [7,8] that the applicability of commonly used HKGs, such as ACTB, GAPDH, and 18sRNA, had crucial limitations. Ideal endogenous HKGs should exhibit consistent expression levels across all experimental conditions (e.g.…”

mentioning

confidence: 99%

Transcriptome-based Selection and Validation of Optimal House-keeping Genes for Skin Research in Goats (Capra hircus)

Zhang

Deng

et al. 2020

Preprint

View full text Add to dashboard Cite

Background: In Quantitative real-time polymerase chain reaction (qRT-PCR) experiments, accurate and reliable target gene expression data is dependent on optimal amplification of house-keeping genes (HKGs). The RNA-seq technology offers a novel approach to detect new HKGs with improved stability. Goat (Capra hircus) is an economically important livestock species, and plays an indispensable role in the world animal fiber and meat industry. Unfortunately, uniform and reliable HKGs be used in skin research of goats have not been identified. Therefore, this study seeks to identify a set of stable HKGs for the skin tissue of C. hircus using the new high-throughput sequencing technology.Results: Based on the transcriptome dataset of 39 goat skin tissues, 8 genes (SRP68, NCBP3, RRAGA, EIF4H, CTBP2, PTPRA, CNBP, and EEF2) with relatively stable expression levels were identified and selected as new candidate HKGs. The classical HKGs including SDHA and YWHAZ from a previous study, and 2 conventional genes (ACTB and GAPDH) were also considered. Four different experimental materials: (1) different development stages, (2) hair follicle cycle stages, (3) breeds and (4) sampling sites were provided for determination and validation. Four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method) and a comprehensive algorithm (ComprFinder, developed in-house) were used to assess the stability of each HKG. It was observed that NCBP3+SDHA+PTPRA was more stably expressed than previously used genes, in all conditions analyzed. This combination was effective at normalizing target gene expression. Moreover, a new algorithm, ComprFinder, was developed and released for comprehensive analysis.Conclusion: This study presents the first data of candidate HKGs selection for skin tissues of C. hircus based on an RNA-seq dataset. We propose the use of the NCBP3+SDHA+PTPRA combination as the triplet HKGs in skin molecular biology in C. hircus and other closely related species in order to standardize analyses across studies. In addition, we also encourage researchers who are performing candidate HKG evaluations and have the needs of a comprehensive analysis to adopt our new algorithm, ComprFinder.

show abstract

Transcriptome‐Based Identification of the Optimal Reference CHO Genes for Normalisation of qPCR Data

Cited by 32 publications

References 38 publications

Transcriptome Analysis of CHO Cell Size Increase During a Fed-Batch Process

Transcriptome Analysis of CHO Cell Size Increase During a Fed-Batch Process

Transcriptome-based Selection and Validation of Optimal House-keeping Genes for Skin Research in Goats (Capra hircus)

Transcriptome-based Selection and Validation of Optimal House-keeping Genes for Skin Research in Goats (Capra hircus)

Contact Info

Product

Resources

About