scholarly journals Genome Wide Search to Identify Reference Genes candidates for Gene Expression Analysis in Gossypium hirsutum

2019 ◽  
Author(s):  
Smitha P K ◽  
Vishnupriyan K ◽  
Ananya S. Kar ◽  
Anil Kumar M ◽  
Christopher Bathula ◽  
...  
Keyword(s):  
Gossypium Hirsutum ◽  
Reference Genes ◽  
Data Science ◽  
Experimental Validation ◽  
Quantitative Polymerase Chain Reaction ◽  
Critical Role ◽  
Genome Wide ◽  
Cotton Species ◽  
Genome Wide Search ◽  
Selection Of

Abstract Background: With the advent of newer breeds and transgenic varieties of commercial crops, qPCR (quantitative polymerase chain reaction) experiments have become extremely popular for quick expression checks. Selection of appropriate reference genes plays a critical role in quantifying the expression of target gene. Most commonly used reference genes in expression studies are the “house-keeping genes”, involved in basic cellular processes. However, expression levels of such genes often vary in response to experimental conditions, forcing the researchers to validate the reference genes in every experiment. This study presents a data science driven unbiased genome-wide search results for selection of reference genes by assessing variation of >50,000 genes in a publicly available RNA-seq dataset of cotton species Gossypium hirsutum. Selected candidate genes were validated experimentally across 33 samples from normal and transgenic G. hirsutum plants, harvested from different areas of the plant at different time points under various developmental conditions. Experimental validation also includes commonly used genes from literature to suggest the most stable set of 5 genes to be used for assessment of quantitative expression in cotton plants (Fig.1). Result: Five genes (TMN5, TBL6, UTR5B, AT1g65240, CYP76B6) identified by data-driven analysis, along with two commonly used reference genes for cotton found in literature (GhPP2A1 and GhuBQ14) were validated using qPCR in a set of 33 experimental samples consisting of different tissues (leaves, square, stem and root), different stages of leaf (young and mature) and square development (small, medium and large) in both transgenic and non-transgenic plants. Expression stability of the genes was evaulated using four different algorithms - DeltaCT, Genorm, BestKeeper and Normfinder. GhPP2A1 and TMN5 were identified as the most stable genes, followed by GhuBQ14 across all the samples tested. Conclusion: This study, for the first time successfully displays a data science driven genome-wide search method followed by experimental validation as a method of choice for selection of stable reference genes for experiment with cotton species. Based on the results we recommend use of GhPP2A1, TMN5 and GhuBQ14 as the optimal candidate reference genes in qPCR experiments with normal or transgenic cotton plant tissues.

scholarly journals Genome Wide Search to Identify Reference Genes candidates for Gene Expression Analysis in Gossypium hirsutum

2019 ◽  
Author(s):  
Smitha P K ◽  
Vishnupriyan K ◽  
Ananya S. Kar ◽  
Anil Kumar M ◽  
Christopher Bathula ◽  
...  
Keyword(s):  
Gossypium Hirsutum ◽  
Reference Genes ◽  
Data Science ◽  
Natural Fiber ◽  
Quantitative Polymerase Chain Reaction ◽  
Critical Role ◽  
Genome Wide ◽  
Cotton Species ◽  
Genome Wide Search ◽  
Selection Of

Abstract Background: Cotton is one of the most important commercial crops as the source of natural fiber, oil and fodder. To protect it from harmful pest populations number of newer transgenic lines have been developed. For quick expression checks in successful agriculture qPCR (quantitative polymerase chain reaction) have become extremely popular. The selection of appropriate reference genes plays a critical role in the outcome of such experiments as the method quantifies expression of the target gene in comparison with the reference. Traditionally most commonly used reference genes are the “ house-keeping genes”, involved in basic cellular processes. However, expression levels of such genes often vary in response to experimental conditions, forcing the researchers to validate the reference genes for every experimental platform. This study presents a data science driven unbiased genome-wide search for the selection of reference genes by assessing variation of >50,000 genes in a publicly available RNA- seq dataset of cotton species Gossypium hirsutum . Result: Five genes ( TMN5, TBL6, UTR5B, AT1g65240 and CYP76B6 ) identified by data-science driven analysis, along with two commonly used reference genes found in literature ( PP2A1 and UBQ14 ) were taken through qPCR in a set of 33 experimental samples consisting of different tissues (leaves, square, stem and root), different stages of leaf (young and mature) and square development (small, medium and large) in both transgenic and non-transgenic plants. Expression stability of the genes was evaluated using four algorithms - geNorm , BestKeeper , NormFinder and RefFinder. Conclusion: Based on the results we recommend the usage of TMN5 and TBL6 as the optimal candidate reference genes in qPCR experiments with normal and transgenic cotton plant tissues. AT1g65240 and PP2A1 can also be used if expression study includes squares. This study, for the first time successfully displays a data science driven genome-wide search method followed by experimental validation as a method of choice for selection of stable reference genes over the selection based on function alone.

scholarly journals Genome wide search to identify reference genes candidates for gene expression analysis in Gossypium hirsutum

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
P. K. Smitha ◽  
K. Vishnupriyan ◽  
Ananya S. Kar ◽  
M. Anil Kumar ◽  
Christopher Bathula ◽  
...  
Keyword(s):  
Gossypium Hirsutum ◽  
Reference Genes ◽  
Data Science ◽  
Natural Fiber ◽  
Quantitative Polymerase Chain Reaction ◽  
Critical Role ◽  
Genome Wide ◽  
Cotton Species ◽  
Genome Wide Search ◽  
Selection Of

Abstract Background Cotton is one of the most important commercial crops as the source of natural fiber, oil and fodder. To protect it from harmful pest populations number of newer transgenic lines have been developed. For quick expression checks in successful agriculture qPCR (quantitative polymerase chain reaction) have become extremely popular. The selection of appropriate reference genes plays a critical role in the outcome of such experiments as the method quantifies expression of the target gene in comparison with the reference. Traditionally most commonly used reference genes are the “house-keeping genes”, involved in basic cellular processes. However, expression levels of such genes often vary in response to experimental conditions, forcing the researchers to validate the reference genes for every experimental platform. This study presents a data science driven unbiased genome-wide search for the selection of reference genes by assessing variation of > 50,000 genes in a publicly available RNA-seq dataset of cotton species Gossypium hirsutum. Result Five genes (TMN5, TBL6, UTR5B, AT1g65240 and CYP76B6) identified by data-science driven analysis, along with two commonly used reference genes found in literature (PP2A1 and UBQ14) were taken through qPCR in a set of 33 experimental samples consisting of different tissues (leaves, square, stem and root), different stages of leaf (young and mature) and square development (small, medium and large) in both transgenic and non-transgenic plants. Expression stability of the genes was evaluated using four algorithms - geNorm, BestKeeper, NormFinder and RefFinder. Conclusion Based on the results we recommend the usage of TMN5 and TBL6 as the optimal candidate reference genes in qPCR experiments with normal and transgenic cotton plant tissues. AT1g65240 and PP2A1 can also be used if expression study includes squares. This study, for the first time successfully displays a data science driven genome-wide search method followed by experimental validation as a method of choice for selection of stable reference genes over the selection based on function alone.

Genome-wide selection of superior reference genes for expression studies in Ganoderma lucidum

Gene ◽  
2015 ◽  
Vol 574 (2) ◽  
pp. 352-358 ◽  
Author(s):  
Zhichao Xu ◽  
Jiang Xu ◽  
Aijia Ji ◽  
Yingjie Zhu ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Reference Genes ◽  
Ganoderma Lucidum ◽  
Expression Studies ◽  
Genome Wide ◽  
Selection Of

scholarly journals Fungal Screening on Olive Oil for Extracellular Triacylglycerol Lipases: Selection of a Trichoderma harzianum Strain and Genome Wide Search for the Genes

Genes ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. 62 ◽  
Author(s):  
Miguel Canseco-Pérez ◽  
Genny Castillo-Avila ◽  
Bartolomé Chi-Manzanero ◽  
Ignacio Islas-Flores ◽  
Max Apolinar-Hernández ◽  
...  
Keyword(s):  
Olive Oil ◽  
Trichoderma Harzianum ◽  
Genome Wide ◽  
Genome Wide Search ◽  
Selection Of

scholarly journals Genome-wide identification of new reference genes for RT-qPCR normalization in CGMMV-infected Lagenaria siceraria

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5642 ◽  
Author(s):  
Chenhua Zhang ◽  
Hongying Zheng ◽  
Xinyang Wu ◽  
Heng Xu ◽  
Kelei Han ◽  
...  
Keyword(s):  
Reference Genes ◽  
Bottle Gourd ◽  
Lagenaria Siceraria ◽  
Rna Seq ◽  
Isopentenyl Transferase ◽  
Genome Wide ◽  
Transcriptome Database ◽  
Qpcr Normalization ◽  
Suitable Reference ◽  
Selection Of

Lagenaria siceraria is an economically important cucurbitaceous crop, but suitable reference genes (RGs) to use when the plants are infected by cucumber green mottle mosaic virus (CGMMV) have not been determined. Sixteen candidate RGs of both leaf and fruit and 18 candidate RGs mostly from separate RNA-Seq datasets of bottle gourd leaf or fruit were screened and assessed by RT-qPCR. The expression stability of these genes was determined and ranked using geNorm, NormFinder, BestKeeper and RefFinder. Comprehensive analysis resulted in the selection of LsCYP, LsH3, and LsTBP as the optimal RGs for bottle gourd leaves, and LsP4H, LsADP, and LsTBP for fruits. LsWD, LsGAPDH, and LsH3 were optimal for use in both leaves and fruits under the infection of CGMMV. Isopentenyl transferase (IPT) and DNA-directed RNA polymerase (DdRP) were used to validate the applicability of the most stable identified RGs from bottle gourd in response to CGMMV. All the candidate RGs performed in RT-qPCR consistently with the data from the transcriptome database. The results demonstrated that LsWD, LsGAPDH and LsH3 were the most suitable internal RGs for the leaf, and LsH3, LsGAPDH, LsP4H and LsCYP for the fruit.

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