Selection of reference genes for qPCR normalization in buffalobur (Solanum rostratum Dunal)

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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Selection of Suitable Reference Genes for qPCR Normalization under Abiotic Stresses and Hormone Stimuli in Carrot Leaves

PLoS ONE ◽

10.1371/journal.pone.0117569 ◽

2015 ◽

Vol 10 (2) ◽

pp. e0117569 ◽

Cited By ~ 90

Author(s):

Chang Tian ◽

Qian Jiang ◽

Feng Wang ◽

Guang-Long Wang ◽

Zhi-Sheng Xu ◽

...

Keyword(s):

Abiotic Stresses ◽

Reference Genes ◽

Qpcr Normalization ◽

Suitable Reference ◽

Selection Of

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Identification of optimal endogenous reference RNAs for RT-qPCR normalization in hindgut of rat models with anorectal malformations

PeerJ ◽

10.7717/peerj.6829 ◽

2019 ◽

Vol 7 ◽

pp. e6829 ◽

Cited By ~ 1

Author(s):

Caiyun Long ◽

Yunxia Xiao ◽

Siying Li ◽

Xiaobing Tang ◽

Zhengwei Yuan ◽

...

Keyword(s):

Reference Genes ◽

Gene Selection ◽

Anorectal Malformations ◽

Limited Data ◽

Rat Fetus ◽

Rat Models ◽

Data Output ◽

Endogenous Reference ◽

Qpcr Normalization ◽

Selection Of

Background Quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive method for quantifying mRNA abundance. With relative expression analysis, however, reliable data output is dependent on stably expressed reference genes across the samples being studied. In anorectal malformations (ARMs), there is limited data on the selection of appropriate reference genes. Purpose This study was aimed to investigate the optimal reference genes for PCR in ARM rat models. Methods We selected 15 commonly used reference genes (Rps18, Actb, B2m, Gapdh, Ppia, Hprt1, Pgk1, Ywhaz, Tbp, Ubc, Rps16, Rpl13a, Rplp1, Sdha, and Hmbs) as candidate reference genes and detected their mRNA expression in ARM samples by RT-qPCR. The expression stability and variability of these transcripts were subsequently evaluated using four methods (geNorm, NormFinder, comparative ΔCt, and BestKeeper). Results The abundance of the candidate reference genes was qualified by RT-qPCR and the cycle threshold (Ct) values ranged between 14.07 (Rplp1) and 21.89 (Sdha). In the overall candidate genes, different variations existed across the different algorithms. A comprehensive analysis revealed that Rpl13a ranked first among the relatively stable genes, followed by Ywhaz, Rps18, Sdha, and Hmbs. Conclusions The most stable reference genes for RT-qPCR were Rpl13a, Ywhaz, and Rps18 in ETU-induced ARMs in rat fetus. This study provided a foundation for reference gene selection for future gene expression analyses.

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Identification and Validation of Reference Genes in Clostridium beijerinckii NRRL B-598 for RT-qPCR Using RNA-Seq Data

Frontiers in Microbiology ◽

10.3389/fmicb.2021.640054 ◽

2021 ◽

Vol 12 ◽

Author(s):

Katerina Jureckova ◽

Hana Raschmanova ◽

Jan Kolek ◽

Maryna Vasylkivska ◽

Barbora Branska ◽

...

Keyword(s):

Reference Genes ◽

Enrichment Analysis ◽

Clostridium Beijerinckii ◽

Stable Expression ◽

Rna Seq ◽

Go Enrichment ◽

Qpcr Normalization ◽

Suitable Reference ◽

Go Enrichment Analysis ◽

Selection Of

Gene expression analysis through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) depends on correct data normalization by reference genes with stable expression. Although Clostridium beijerinckii NRRL B-598 is a promising Gram-positive bacterium for the industrial production of biobutanol, validated reference genes have not yet been reported. In this study, we selected 160 genes with stable expression based on an RNA sequencing (RNA-Seq) data analysis, and among them, seven genes (zmp, rpoB1, rsmB, greA, rpoB2, topB2, and rimO) were selected for experimental validation by RT-qPCR and gene ontology (GO) enrichment analysis. According to statistical analyses, zmp and greA were the most stable and suitable reference genes for RT-qPCR normalization. Furthermore, our methodology can be useful for selection of the reference genes in other strains of C. beijerinckii and it also suggests that the RNA-Seq data can be used for the initial selection of novel reference genes, however, their validation is required.

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