scholarly journals Evaluation of qPCR Reference Genes For Taimen (Hucho Taimen)

2021 ◽  
Author(s):  
Xiaoxing Yang ◽  
Guangxiang Tong ◽  
Le Dong ◽  
Guopan Tang ◽  
Yongquan Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Reference Genes ◽  
28S Rrna ◽  
Experimental Conditions ◽  
Stress Genes ◽  
Qrt Pcr ◽  
Processing Times ◽  
Reliable Quantification ◽  
Hucho Taimen

Abstract As a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquacultural research. Understanding the biology of taimen (Hucho taimen) has attracted increased interest because of its ecological and economic values. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimised for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using four methods, geNorm, NormFinder, BestKeeper and RefFinder. The expression levels of the 10 genes were detected using samples from 48 experimental groups that consisted of six tissues (blood, heart, brain, gill, skin and liver) collected under five heat-stress conditions (18, 20, 22, 24 and 26°C) and four heat-stress processing times (4, 24, 48 and 96 h). RPS29 and RPL19 were the most stable genes among all the samples, whereas 28S rRNA, ARBPR and 18S rRNA were the least stable. These results were verified by an expression analysis of the heat-stress genes (hsp60 and hsp70) of taimen. In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions, and these results allow the reliable study of gene expression in taimen.

scholarly journals Evaluation of qPCR reference genes for taimen (Hucho taimen) under heat stress

2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaoxing Yang ◽  
Guangxiang Tong ◽  
Le Dong ◽  
Ting Yan ◽  
Huan Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Ribosomal Protein ◽  
Ribosomal Rna ◽  
Reference Genes ◽  
28S Rrna ◽  
Qrt Pcr ◽  
Hucho Taimen

AbstractAs a powerful and attractive method for detecting gene expression, qRT-PCR has been broadly used in aquaculture research. Understanding the biology of taimen (Hucho taimen) has drawn increasing interest because of its ecological and economic value. Stable reference genes are required for the reliable quantification of gene expression, but such genes have not yet been optimized for taimen. In this study, the stability levels of 10 commonly used candidate reference genes were evaluated using geNorm, NormFinder, BestKeeper, and RefFinder. The expression levels of the 10 genes were detected using 240 samples from 48 experimental groups consisting of 40 individuals treated under four heat-stress conditions (18, 20, 22, and 24 °C) for 24 h and 26 °C for 4, 24, 48, and 72 h. Six tissues (blood, heart, brain, gill, skin, and liver) were collected from each individual. Ribosomal protein S29 (RPS29) and ribosomal protein L19 (RPL19) were the most stable genes among all of the samples, whereas 28S ribosomal RNA (28S rRNA), attachment region binding protein (ARBP), and 18S ribosomal RNA (18S rRNA) were the least stable. These results were verified by an expression analysis of taimen heat-stress genes (heat shock protein 60, hsp60, and heat shock protein 70, hsp70). In conclusion, RPS29 and RPL19 are the optimal reference genes for qRT-PCR analyses of taimen, irrespective of the tissue and experimental conditions. These results allow the reliable study of gene expression in taimen.

scholarly journals Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Forests ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 193
Author(s):  
Dandan Li ◽  
Sen Yu ◽  
Minzhen Zeng ◽  
Xiao Liu ◽  
Jia Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Real Time ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Stable Gene ◽  
Larix Olgensis ◽  
Qrt Pcr ◽  
Study Gene Expression ◽  
Selection Of

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

scholarly journals Evaluation of Optimal Reference Genes for qRT-PCR Analysis in Hyphantria cunea (Drury)

Insects ◽  
2022 ◽  
Vol 13 (1) ◽  
pp. 97
Author(s):  
Xudong Zhao ◽  
Yishu Geng ◽  
Tianyi Hu ◽  
Yongang Zhao ◽  
Suling Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Comprehensive Evaluation ◽  
Larval Density ◽  
Physiological Role ◽  
Invasive Pest ◽  
Pcr Analysis ◽  
Hyphantria Cunea ◽  
Experimental Conditions ◽  
Qrt Pcr

The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, β-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species.

scholarly journals Selection and validation of experimental condition-specific reference genes for qRT-PCR in Metopolophium dirhodum (Walker) (Hemiptera: Aphididae)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Xinan Li ◽  
Peipan Gong ◽  
Bingting Wang ◽  
Chao Wang ◽  
Mengyi Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Body Part ◽  
Specific Reference ◽  
Expression Stability ◽  
Experimental Conditions ◽  
Metopolophium Dirhodum ◽  
Geographic Population ◽  
Qrt Pcr ◽  
Winter Cereals

AbstractMetopolophium dirhodum (Walker) (Hemiptera: Aphididae) is one of the most common aphid pests of winter cereals. To facilitate accurate gene expression analyses with qRT-PCR assays, the expression stability of candidate reference genes under specific experimental conditions must be verified before they can be used to normalize target gene expression levels. In this study, 10 candidate reference genes in M. dirhodum were analyzed by qRT-PCR under various experimental conditions. Their expression stability was evaluated with delta Ct, BestKeeper, geNorm, and NormFinder methods, and the final stability ranking was determined with RefFinder. The results indicate that the most appropriate sets of internal controls were SDHB and RPL8 across geographic population; RPL8, Actin, and GAPDH across developmental stage; SDHB and NADH across body part; RPL8 and Actin across wing dimorphism and temperature; RPL4 and EF1A across starvation stress; AK and RPL4 across insecticide treatments; RPL8 and NADH across antibiotic treatments; RPL8, RPL4, Actin, and NADH across all samples. The results of this study provide useful insights for establishing a standardized qRT-PCR procedure for M. dirhodum and may be relevant for identifying appropriate reference genes for molecular analyses of related insects.

scholarly journals Validation of reference genes for quantitative RT-PCR normalization inSuaeda aralocaspica, an annual halophyte with heteromorphism and C4 pathway without Kranz anatomy

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1697 ◽  
Author(s):  
Jing Cao ◽  
Lu Wang ◽  
Haiyan Lan
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Transcriptional Level ◽  
28S Rrna ◽  
Related Gene ◽  
Kranz Anatomy ◽  
Qrt Pcr ◽  
C4 Pathway ◽  
The Stability

Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful analytical technique for the measurement of gene expression, which depends on the stability of the reference gene used for data normalization.Suaeda aralocaspica, an annual halophyte with heteromorphic seeds and possessing C4 photosynthesis pathway without Kranz anatomy, is an ideal plant species to identify stress tolerance-related genes and compare relative expression at transcriptional level. So far, no molecular information is available for this species. In the present study, six traditionally used reference genes were selected and their expression stability in two types of seeds ofS. aralocaspicaunder different experimental conditions was evaluated. Three analytical programs, geNorm, NormFinder and BestKeeper, were used to assess and rank the stability of reference gene expression. Results revealed that although some reference genes may display different transcriptional profiles between the two types of seeds,β-TUB andGAPDHappeared to be the most suitable references under different developmental stages and tissues.GAPDHwas the appropriate reference gene under different germination time points and salt stress conditions, andACTINwas suitable for various abiotic stress treatments for the two types of seeds. For all the sample pools,β-TUB served as the most stable reference gene, whereas18S rRNAand28S rRNAperformed poorly and presented as the least stable genes in our study.UBQseemed to be unsuitable as internal control under different salt treatments. In addition, the expression of a photosynthesis-related gene (PPDK) of C4 pathway and a salt tolerance-related gene (SAT) ofS. aralocaspicawere used to validate the best performance reference genes. This is the first systematic comparison of reference gene selection for qRT-PCR work inS. aralocaspicaand these data will facilitate further studies on gene expression in this species and other euhalophytes.

scholarly journals Evaluation of Reference Genes for qRT-PCR Normalization in Angelica decursiva under Various Experimental Conditions

2020 ◽  
Author(s):  
Yuedong He ◽  
Yuan Zhong ◽  
Zhenzhen Bao ◽  
Weiqi Wang ◽  
Xiaoqing Xu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Binding Protein ◽  
Pcr Analysis ◽  
Experimental Conditions ◽  
Angelica Decursiva ◽  
Qrt Pcr ◽  
Polypyrimidine Tract Binding Protein ◽  
Chinese Medicinal Plants ◽  
The Stability

Angelica decursiva is one of the lending traditional Chinese medicinal plants producing coumarins. Notably, several studies have focused on the biosynthesis and not the qRT-PCR (quantitative real-time reverse transcription polymerase chain reaction) study of coumarins. This qRT-PCR technique has been extensively used to investigate gene expression levels in plants and the selection of reference genes which plays a crucial role in standardizing the data form the qRT-PCR analysis. In our study, 11 candidate reference genes were selected from the existing transcriptome data of Angelica decursiva. Here, four different types of statistical algorithms (geNorm, NormFinder, BestKeeper, and Delta Ct) were used to calculate and evaluate the stability of gene expression under different external treatments. Subsequently, RefFinder analysis was used to determine the geometric average of each candidate gene ranking, and to perform comprehensive index ranking. The obtained results showed that among all the 11 candidate reference genes, SAND family protein (SAND), protein phosphatase 2A gene (PP2A), and polypyrimidine tract-binding protein (PTBP) were the most stable reference genes, where Nuclear cap binding protein 2 (NCBP2), TIP41-like protein (TIP41), and Beta-6-tubulin (TUBA) were the least stable genes. To the best of our knowledge, this work is the first to evaluate the stability of reference genes in the Angelica decursiva which has provided an important foundation on the use of qRT-PCR for an accurate and far-reaching gene expression analysis in this medicinal plant.

scholarly journals Selection and validation of reference genes for quantitative gene expression normalization in Taxus spp.

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Kaikai Zhang ◽  
Wei Fan ◽  
Duanfen Chen ◽  
Luyuan Jiang ◽  
Yunfeng Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Reference Genes ◽  
Expression Patterns ◽  
Future Research ◽  
Taxus Chinensis ◽  
Experimental Conditions ◽  
Taxol Biosynthesis ◽  
Qrt Pcr ◽  
Suitable Reference

AbstractQuantitative real-time PCR (qRT-PCR) is commonly used to measure gene expression to further explore gene function, while suitable reference genes must be stably expressed under different experimental conditions to obtain accurate and reproducible data for relative quantification. Taxol or paclitaxel is an important anticancer compound mainly identified in Taxus spp. The molecular mechanism of the regulation of taxol biosynthesis is current research goal. However, in the case of Taxus spp., few reports were published on screening suitable reference genes as internal controls for qRT-PCR. Here, eight reference genes were selected as candidate reference genes for further study. Common statistical algorithms geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to analyze the data from samples collected from a cell line of Taxus × media under various experimental conditions and from tissues of Taxus chinensis var. mairei. The expression patterns of TcMYC under salicylic acid treatment differed significantly, with the best and worst reference genes in the cell line. This study screened out suitable reference genes (GAPDH1 and SAND) under different treatments and tissues for the accurate and reliable normalization of the qRT-PCR expression data of Taxus spp. At the same time, this study will aid future research on taxol biosynthesis-related genes expression in Taxus spp., and can also be directly used to other related species.

scholarly journals Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Won-Jae Lee ◽  
Ryoung-Hoon Jeon ◽  
Si-Jung Jang ◽  
Ji-Sung Park ◽  
Seung-Chan Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Optimal Number ◽  
Experimental Conditions ◽  
Pearson’S Correlation ◽  
Qrt Pcr ◽  
Differentiated Cells ◽  
Pearson's Correlation ◽  
Suitable Reference

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson’s correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson’s correlation revealed high correlation (r>0.9) between NF3and NFopt. Overall, the present study showed thatHMBS,YWHAZ,SDHA, andTBPare suitable reference genes for qRT-PCR in porcine MSCs.

scholarly journals Validation of Reference Genes for Studying Different Abiotic Stresses in oat (Avena sativa L.) by RT-qPCR

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1272
Author(s):  
Judit Tajti ◽  
Magda Pál ◽  
Tibor Janda
Keyword(s):  
Gene Expression ◽  
Avena Sativa ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Nutritional Value ◽  
Tissue Type ◽  
Future Research ◽  
Experimental Conditions ◽  
Expression Studies ◽  
Gene Expression Studies

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

scholarly journals Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 960
Author(s):  
Meagan Archer ◽  
Jianping Xu
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Specific Method ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Physiological Processes ◽  
The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

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