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

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6829 ◽  
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.

scholarly journals Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone Flaccida Fr. Schmidt at Different Growing Stages

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 459
Author(s):  
Zeying Zhao ◽  
Hanwen Zhou ◽  
Zhongnan Nie ◽  
Xuekui Wang ◽  
Biaobiao Luo ◽  
...  
Keyword(s):  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Cultivated Plants ◽  
Gene Expressions ◽  
Natural Habitats ◽  
Triterpenoid Saponins ◽  
Qpcr Normalization ◽  
Suitable Reference

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

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

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dandan Zhao ◽  
Xu Wang ◽  
Jingchao Chen ◽  
Zhaofeng Huang ◽  
Heqiang Huo ◽  
...  
Keyword(s):  
Reference Genes ◽  
Solanum Rostratum ◽  
Qpcr Normalization ◽  
Selection Of

scholarly journals ERgene: Python library for screening endogenous reference genes

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Zehua Zeng ◽  
Yuzhe Xiong ◽  
Wenhuan Guo ◽  
Hongwu Du
Keyword(s):  
Reference Genes ◽  
High Efficiency ◽  
Reference Method ◽  
Internal Reference ◽  
Matrix Block ◽  
Endogenous Genes ◽  
Endogenous Reference ◽  
High Computational Efficiency ◽  
Selection Of ◽  
Endogenous Reference Genes

Abstract In gene expression analysis, sample differences and experimental operation differences are common, but sometimes, these differences will cause serious errors to the results or even make the results meaningless. Finding suitable internal reference genes efficiently to eliminate errors is a challenge. Aside from the need for high efficiency, there is no package for screening endogenous genes available in Python. Here, we introduce ERgene, a Python library for screening endogenous reference genes. It has extremely high computational efficiency and simple operation steps. The principle is based on the inverse process of the internal reference method, and the robust matrix block operation makes the selection of internal reference genes faster than any other method.

scholarly journals Selection of Suitable Reference Genes for qPCR Normalization under Abiotic Stresses in Oenanthe javanica (BI.) DC

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e92262 ◽  
Author(s):  
Qian Jiang ◽  
Feng Wang ◽  
Meng-Yao Li ◽  
Jing Ma ◽  
Guo-Fei Tan ◽  
...  
Keyword(s):  
Abiotic Stresses ◽  
Reference Genes ◽  
Oenanthe Javanica ◽  
Qpcr Normalization ◽  
Suitable Reference ◽  
Selection Of

scholarly journals RNA-Seq is not required to determine stable reference genes for qPCR normalization

2021 ◽  
Author(s):  
Nirmal Kumar Sampathkumar ◽  
Venkat Krishnan Sundaram ◽  
Prakroothi S Danthi ◽  
Rasha Barakat ◽  
Shiden Solomon ◽  
...  
Keyword(s):  
Reference Genes ◽  
Statistical Approach ◽  
Gene Selection ◽  
Data Normalization ◽  
Rna Seq ◽  
Experimental Conditions ◽  
Cross Sectional ◽  
Qpcr Data ◽  
Statistical Approaches ◽  
Qpcr Normalization

AbstractAssessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes. Although numerous statistical approaches have been proposed to determine the best reference genes, they can give rise to conflicting results depending on experimental conditions. Hence, recent studies propose the use of RNA-Seq to identify stable genes followed by the application of different statistical approaches to determine the best set of reference genes for qPCR data normalization. In this study, we demonstrate that the statistical approach to determine the best reference genes from randomly selected candidates is more important than the preselection of ‘stable’ candidates from RNA-Seq data. Using a qPCR data normalization workflow that we have previously established; we show that qPCR data normalization using randomly chosen conventional reference genes renders the same results as stable reference genes selected from RNA-Seq data. We validated these observations in two distinct cross-sectional experimental conditions involving human iPSC derived microglial cells and mouse sciatic nerves. These results taken together show that given a robust statistical approach for reference gene selection, stable genes selected from RNA-Seq data do not offer any significant advantage over commonly used reference genes for normalizing qPCR assays.

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