scholarly journals Selection of Reference Genes for Normalization of Real-Time PCR Data in Calliptamus italicus (Orthoptera: Acrididae) Under Different Temperature Conditions

2019 ◽  
Vol 19 (6) ◽  
Author(s):  
Hongxia Hu ◽  
Xiaofang Ye ◽  
Han Wang ◽  
Rong Ji
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Analysis ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Target Gene ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Target Gene Expression

Abstract Global warming has dominated worldwide climate change trends, and adaptability to high temperatures is the main factor underlying the spread of the pest Calliptamus italicus in Xinjiang Province, China. However, knowledge about the molecular mechanisms responsible for this adaptability and other related biological properties of C. italicus remain relatively unclear. Real-time quantitative polymerase chain reaction (RT-qPCR) is a key tool for gene expression analysis associated with various biological processes. Reference genes are necessary for normalizing gene expression levels across samples taken from specific experimental conditions. In this study, transcript level of five genes (GAPDH, 18S, TUB, ACT, and EF1α), commonly used as reference genes, were evaluated under nine different temperatures (27, 30, 33, 36, 39, 42, 45, 48, and 51°C) to assess their expression stability and further select the most suitable to be used on normalization of target gene expression data. Gene expression profiles were analyzed using geNorm, NormFinder, and BestKeeper software packages. The combined results demonstrated that the best-ranked reference genes for C. italicus are EF1α, GAPDH, and ACT under different thermal stress conditions. This is the first study that assesses gene expression analysis across a range of temperatures to select the most appropriate reference genes for RT-qPCR data normalization in C. italicus. These results should assist target gene expression analysis associated with heat stress in C. italicus.

scholarly journals Identification and Evaluation of Reference Genes for Normalization of Gene Expression in Developmental Stages, Sexes, and Tissues of Diaphania caesalis (Lepidoptera, Pyralidae)

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zheng Wang ◽  
Qianqian Meng ◽  
Xi Zhu ◽  
Shiwei Sun ◽  
Aiqin Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Target Gene ◽  
Target Genes ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Transcriptional Level ◽  
Internal Reference ◽  
Target Gene Expression ◽  
Qrt Pcr

Abstract Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, β-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, β-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.

scholarly journals Selection and Validation of Reference Genes for Quantitative Real-Time PCR Expression Analysis of Candidate Genes in Carposina sasakii (Lepidoptera: Carposinidae)

2020 ◽  
pp. 75-85
Author(s):  
Zuobing Yan ◽  
Yongli Li ◽  
Zhou Zhou ◽  
Yongan Zhang ◽  
Liangjian Qu
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Analysis ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Quantitative Real Time Pcr ◽  
Carposina Sasakii

Carposina sasakii is one of the most important pests on the quality of stone and pome fruits. Investigation of a gene expression level in the species is hampered because of the gap of validated reference genes. The expression variation in the transcription levels of eight candidate reference genes, Actin (ACT), Tubulinbeta-1 (TUB), Ribosomal protein 49 (RP49), Elongation factor1-alpha (EF-1a), Elongation factor1-b (EF-1b), Elongation factor1-d (EF-1d), Ribosomal proteinL13 (RPL13) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were analyzed by quantitative real-time PCR (qPCR). The stability and ranking of these gene expression profiles in three organ types (head, thorax and abdomen), three developmental stages (larva, pupa and moth), and five diapause states (non-diapause, pre-diapause, diapause 0 d, diapause 20 d and diapause 60 d) were assessed using two algorithm-based methods, geNorm and NormFinder. EF-1a, ACT and GAPDH were evaluated to be the three stable reference genes based on the important observations and comprehensive analysis, whereas TUB and EF-1b showed low expression stability. Best gene combinations for different qPCR analysis in C. sasakii could be chosen from the three stable reference genes, the using of two reference genes is sufficient to effectively normalize qPCR data in C. sasakii. The study laid the foundation for gene expression analysis in C. sasakii and provided new information for the selection of reference genes.

scholarly journals Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal

2018 ◽  
Vol 18 (1) ◽  
Author(s):  
Rikki A. M. Brown ◽  
Michael R. Epis ◽  
Jessica L. Horsham ◽  
Tasnuva D. Kabir ◽  
Kirsty L. Richardson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Target Gene ◽  
Rna Extraction ◽  
Total Rna ◽  
Target Gene Expression ◽  
Total Rna Extraction

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses

2019 ◽  
Vol 46 (2) ◽  
pp. 1809-1817 ◽  
Author(s):  
Pawan Shukla ◽  
Ramesha A. Reddy ◽  
Kangayam M. Ponnuvel ◽  
Gulab Khan Rohela ◽  
Aftab A. Shabnam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Expression Analysis ◽  
Real Time Pcr ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Quantitative Real Time Pcr ◽  
Suitable Reference ◽  
Selection Of
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