scholarly journals Identification of Reference Genes for Expression Studies in the Whole-Blood from Three Cattle Breeds under Two States of Livestock Weather Safety

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3073
Author(s):  
Kelly J. Lozano-Villegas ◽  
Roy Rodríguez-Hernández ◽  
María P. Herrera-Sánchez ◽  
Heinner F. Uribe-García ◽  
Juan S. Naranjo-Gómez ◽  
...  
Keyword(s):  
Whole Blood ◽  
Reference Genes ◽  
18S Rrna ◽  
Relative Quantification ◽  
Experimental Conditions ◽  
Cattle Breeds ◽  
Expression Studies ◽  
Safety Alert ◽  
Lower Variation ◽  
The Stability

Real-time PCR is widely used to study the relative abundance of mRNA due to its specificity, sensitivity, and repeatability quantification. However, relative quantification requires a reference gene, which should be stable in its expression, showing lower variation by experimental conditions or tissues. The aim of this study was to evaluate the stability of the expression of five commonly used reference genes (actb, ywhaz, b2m, sdha, and 18s rRNA) at different physiological stages (alert and emergency) in three different cattle breeds. In this study, five genes (actb, ywhaz, b2m, sdha, and 18s rRNA) were selected as candidate reference genes for expression studies in the whole blood from three cattle breeds (Romosinuano, Gyr, and Brahman) under heat stress conditions. The transcription stability of the candidate reference genes was evaluated using geNorm and NormFinder. The results showed that actb, 18SrRNA, and b2m expression were the most stable reference genes for whole blood of Gyr and Brahman breeds under two states of livestock weather safety (alert and emergency). Meanwhile, actb, b2m, and ywhaz were the most stable reference genes for the Romosinuano breed.

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.

scholarly journals Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dorota M. Krzyżanowska ◽  
Anna Supernat ◽  
Tomasz Maciąg ◽  
Marta Matuszewska ◽  
Sylwia Jafra
Keyword(s):  
Gene Expression ◽  
Quorum Sensing ◽  
Reference Genes ◽  
Significance Threshold ◽  
Expression Studies ◽  
Reverse Transcription Quantitative Pcr ◽  
Gene Expression Studies ◽  
The Stability ◽  
Selection Of

Abstract Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

scholarly journals Validation of Internal Control Genes for Quantitative Real-Time PCR Gene Expression Analysis in Morchella

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2331 ◽  
Author(s):  
Qianqian Zhang ◽  
Wei Liu ◽  
Yingli Cai ◽  
A-Feng Lan ◽  
Yinbing Bian
Keyword(s):  
Gene Expression ◽  
Candidate Genes ◽  
Expression Analysis ◽  
Internal Control ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Stable Gene ◽  
Experimental Conditions ◽  
The Stability ◽  
Vacuolar Protein

The reliability of qRT-PCR results depend on the stability of reference genes used for normalization, suggesting the necessity of identification of reference genes before gene expression analysis. Morels are edible mushrooms well-known across the world and highly prized by many culinary kitchens. Here, several candidate genes were selected and designed according to the Morchella importuna transcriptome data. The stability of the candidate genes was evaluated with geNorm and NormFinder under three different experimental conditions, and several genes with excellent stability were selected. The extensive adaptability of the selected genes was tested in ten Morchella species. Results from the three experimental conditions revealed that ACT1 and INTF7 were the most prominent genes in Morchella, CYC3 was the most stable gene in different development stages, INTF4/AEF3 were the top-ranked genes across carbon sources, while INTF3/CYC3 pair showed the robust stability for temperature stress treatment. We suggest using ACT1, AEF3, CYC3, INTF3, INTF4 and INTF7 as reference genes for gene expression analysis studies for any of the 10 Morchella strains tested in this study. The stability and practicality of the gene, vacuolar protein sorting (INTF3), vacuolar ATP synthase (INTF4) and14-3-3 protein (INTF7) involving the basic biological processes were validated for the first time as the candidate reference genes for quantitative PCR. Furthermore, the stability of the reference genes was found to vary under the three different experimental conditions, indicating the importance of identifying specific reference genes for particular conditions.

scholarly journals Selecting and validating reference genes for quantitative real-time PCR in Plutella xylostella (L.)

Genome ◽  
2018 ◽  
Vol 61 (5) ◽  
pp. 349-358 ◽  
Author(s):  
Yanchun You ◽  
Miao Xie ◽  
Liette Vasseur ◽  
Minsheng You
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Plutella Xylostella ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Real Time Pcr ◽  
Experimental Conditions ◽  
Expression Studies ◽  
Gene Expression Studies

Gene expression analysis provides important clues regarding gene functions, and quantitative real-time PCR (qRT-PCR) is a widely used method in gene expression studies. Reference genes are essential for normalizing and accurately assessing gene expression. In the present study, 16 candidate reference genes (ACTB, CyPA, EF1-α, GAPDH, HSP90, NDPk, RPL13a, RPL18, RPL19, RPL32, RPL4, RPL8, RPS13, RPS4, α-TUB, and β-TUB) from Plutella xylostella were selected to evaluate gene expression stability across different experimental conditions using five statistical algorithms (geNorm, NormFinder, Delta Ct, BestKeeper, and RefFinder). The results suggest that different reference genes or combinations of reference genes are suitable for normalization in gene expression studies of P. xylostella according to the different developmental stages, strains, tissues, and insecticide treatments. Based on the given experimental sets, the most stable reference genes were RPS4 across different developmental stages, RPL8 across different strains and tissues, and EF1-α across different insecticide treatments. A comprehensive and systematic assessment of potential reference genes for gene expression normalization is essential for post-genomic functional research in P. xylostella, a notorious pest with worldwide distribution and a high capacity to adapt and develop resistance to insecticides.

scholarly journals Identification and Validation of Reference Genes for Gene Expression Analysis in Different Development Stages of Amylostereum areolatum

2022 ◽  
Vol 12 ◽  
Author(s):  
Ningning Fu ◽  
Jiaxing Li ◽  
Ming Wang ◽  
Lili Ren ◽  
Shixiang Zong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Growth And Development ◽  
Target Genes ◽  
Quantitative Polymerase Chain Reaction ◽  
Growth Stages ◽  
Experimental Conditions ◽  
Development Stages ◽  
Symbiotic Fungus ◽  
The Stability

A strict relationship exists between the Sirex noctilio and the Amylostereum areolatum, which is carried and spread by its partner. The growth and development of this symbiotic fungus is key to complete the life history of the Sirex woodwasp. Real-time quantitative polymerase chain reaction (RT-qPCR) is used to measure gene expression in samples of A. areolatum at different growth stages and explore the key genes and pathways involved in the growth and development of this symbiotic fungus. To obtain accurate RT-qPCR data, target genes need to be normalized by reference genes that are stably expressed under specific experimental conditions. In our study, the stability of 10 candidate reference genes in symbiotic fungal samples at different growth and development stages was evaluated using geNorm, NormFinder, BestKeeper, delta Ct methods, and RefFinder. Meanwhile, laccase1 was used to validate the stability of the selected reference gene. Under the experimental conditions of this study, p450, CYP, and γ-TUB were identified as suitable reference genes. This work is the first to systematically evaluate the reference genes for RT-qPCR results normalization during the growth of this symbiotic fungus, which lays a foundation for further gene expression experiments and understanding the symbiotic relationship and mechanism between S. noctilio and A. areolatum.

scholarly journals Development of Sarcophaga dux (Diptera: Sarcophagidae) at constant temperatures and differential gene expression for age estimation of intrapuparial

2019 ◽  
Author(s):  
Xiangyan Zhang ◽  
Yi Li ◽  
Yanjie Shang ◽  
Lipin Ren ◽  
Wei Chen ◽  
...  
Keyword(s):  
Age Estimation ◽  
Reference Genes ◽  
18S Rrna ◽  
Differentially Expressed Gene ◽  
Differentially Expressed ◽  
Threshold Temperature ◽  
28S Rrna ◽  
Experimental Conditions ◽  
Developmental Threshold ◽  
Developmental Models

Abstract BackgroundSarcophaga dux (Diptera: Sarcophagidae) is a necrophagous flesh fly with potential forensic value in estimating minimum postmortem interval (PMImin). The basic developmental data and precise intrapuparial age estimates are significant for PMImin estimationof application entomological data in legal medicine.MethodsThe development parameters of S. dux at seven constant temperatures from 16°C to 34°C were investigated by rearing using pig lung in the artificial climate box. The appropriate reference genes and intrapuparial differentially expressed genes (DEGs) of S. dux at constant temperatures 34, 25 and 16°C were selected and analyzed using RT-qPCR for more precisely age estimations. ResultsThe developmental durations of S. dux at 16, 19, 22, 25, 28, 31 and 34°C from larviposition to adult eclosion were 1478.6±18.3, 726.1±15.8, 538.5±0.9, 394.1±9.5, 375.6±10.8, 284.1±7.3, and 252.5±6.1 h, respectively. The thermal summation constant of S. dux was 5341.71±249.29 degree hours, and the developmental threshold temperature was 12.266±0.35°C. The most reliable reference genes under intrapuparial and different temperature in our study were: GST1 and 18S rRNA for 34°C group, GST1 and RPL49 for 25 °C group, and 18S rRNA and 28S rRNA for 16 °C group，the four differential expression genes (Hsp60, A-alpha, ARP, RPL8) can be used to more precisely intrapuparial age estimation of S. dux. ConclusionsThe basic developmental data of S. dux at constant temperatures, such as body length changing of the larva period, accumulated degree hours and duration of development, to establish developmental models that can be used to estimate the PMImin. The selection and evaluation of appropriate reference genes at different experimental conditions are primary with RT-qPCR. The differentially expressed gene can contribute to more accuracy age estimations of S. dux intrapuparial. The result from this study can make contributes to the use of S.dux for estimating PMImin.

scholarly journals Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Hao Xie ◽  
Bo Li ◽  
Yu Chang ◽  
Xiaoyan Hou ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Reference Genes ◽  
18S Rrna ◽  
Spinacia Oleracea ◽  
Expression Patterns ◽  
Gene Expression Pattern ◽  
Housekeeping Genes ◽  
Qpcr Analysis ◽  
The Stability

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl2, NaCl, NaHCO3, and Na2CO3 stresses. The expression of these genes was quantified by RT-qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat-responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT-qPCR analysis.

scholarly journals Evaluation of Reference Genes and Normalization Strategy for Quantitative Real-Time PCR in Human Pancreatic Carcinoma

2012 ◽  
Vol 32 (3) ◽  
pp. 203-210 ◽  
Author(s):  
Beatrice Mohelnikova-Duchonova ◽  
Martin Oliverius ◽  
Eva Honsova ◽  
Pavel Soucek
Keyword(s):  
Pancreatic Carcinoma ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Rna Degradation ◽  
Pancreatic Tumors ◽  
Quantitative Real Time Pcr ◽  
Expression Studies ◽  
The Stability ◽  
Suitable Reference

Histologically verified pairs (n= 10) of pancreatic tumors and non-neoplastic tissues were used for quantitative real-time PCR and the stability of 24 reference genes was analyzed with geNorm and NormFinder software. Raw Cqvalues correlated with the degree of RNA degradation. This correlation was abolished by normalization to Cqof 18S endogenous control gene. Both geNorm and NormFinder programs suggested EIF2B1, ELF1, MRPL19, and POP4 as the same most stable genes. We have thus identified suitable reference genes for future expression studies in pancreatic carcinoma. Normalization method reducing the effects of RNA degradation on the quality of results was also developed.

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