Identification and validation of reference genes for qRT-PCR based studies in horse gram (Macrotyloma uniflorum)

Sinha, Ragini; Bala, Meenu; Prabha, Pragya; Ranjan, Alok; Chahota, Rakesh Kumar; Sharma, T. R.; Singh, Anil Kumar

doi:10.1007/s12298-021-01104-0

Cited by 3 publications

(1 citation statement)

References 63 publications

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“…Gene expression analysis is valuable for obtaining information about cellular mechanisms in different biological samples [ 1 ]. Quantitative real-time PCR (RT-qPCR) is a widely used technique to access the gene expression of a specific gene.…”

Section: Introductionmentioning

confidence: 99%

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR

de Brito,

de Carvalho,

Mota

et al. 2024

BMC Genomics

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Background Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. Results We developed a software named “Gene Selector for Validation” (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. Conclusion The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data.

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Section: Introductionmentioning

confidence: 99%

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR

de Brito,

de Carvalho,

Mota

et al. 2024

BMC Genomics

View full text Add to dashboard Cite

show abstract

Horse Gram (Macrotyloma uniflorum(Lam.) Verdc.)

Philanim,

Kumar,

Bhupenchandra

et al. 2024

Potential Pulses

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Selection of Reference Genes in Siraitia siamensis and Expression Patterns of Genes Involved in Mogrosides Biosynthesis

Chen,

Lin,

Wang

et al. 2024

Plants

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Siraitia siamensis is a traditional Chinese medicinal herb. In this study, using S. siamensis cultivated in vitro, twelve candidate reference genes under various treatments were analyzed for their expression stability by using algorithms such as GeNorm, NormFinder, BestKeeper, Delta CT, and RefFinder. The selected reference genes were then used to characterize the gene expression of cucurbitadienol synthase, which is a rate-limiting enzyme for mogroside biosynthesis. The results showed that CDC6 and NCBP2 expression was the most stable across all treatments and are the best reference genes under the tested conditions. Utilizing the validated reference genes, we analyzed the expression profiles of genes related to the synthesis pathway of mogroside in S. siamensis in response to a range of abiotic stresses. The findings of this study provide clear standards for gene expression normalization in Siraitia plants and exploring the rationale behind differential gene expression related to mogroside synthesis pathways.

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Identification and validation of reference genes for qRT-PCR based studies in horse gram (Macrotyloma uniflorum)

Cited by 3 publications

References 63 publications

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR

RNA-seq validation: software for selection of reference and variable candidate genes for RT-qPCR

Horse Gram (Macrotyloma uniflorum(Lam.) Verdc.)

Selection of Reference Genes in Siraitia siamensis and Expression Patterns of Genes Involved in Mogrosides Biosynthesis

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