scholarly journals The analysis of reference genes expression stability in susceptible and resistant Apera spica-venti populations under herbicide treatment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Barbara Wrzesińska ◽  
Karolina Kościelniak ◽  
Patryk Frąckowiak ◽  
Tadeusz Praczyk ◽  
Aleksandra Obrępalska-Stęplowska
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Crop Production ◽  
Expression Profiles ◽  
Housekeeping Genes ◽  
Resistance Mechanisms ◽  
Target Site ◽  
Expression Stability ◽  
Target Site Resistance ◽  
Tata Box Binding Protein

AbstractWeed resistance to herbicides constitutes a serious problem to world crop production. One of the weeds that are significantly threatening the crops’ yield and quality is Apera spica-venti. The target-site resistance (TSR) mechanism of A. spica-venti has been widely studied, though, little is known about its non-target-site resistance (NTSR) mechanisms at the molecular level. Molecular examination of NTSR is, to a great extent, based on the expression profiles of selected genes, e.g. those participating in detoxification. However, to obtain reliable results of gene expression analysis, the use of a normalizer is required. The aim of this study was to select the best reference genes in A. spica-venti plants of both populations, susceptible and resistant to ALS inhibitor, under treatment with herbicide. Eleven housekeeping genes were chosen for their expression stability assessment. The efficiency correction of raw quantification cycles (Cq) was included in the gene expression stability analyses, which resulted in indicating the TATA-box binding protein (TBP), glyceraldehyde-3-phosphate dehydrogenase, cytosolic (GAPC), and peptidyl-prolyl cis–trans isomerase CYP28 (CYP28) genes as the most stably expressed reference genes. The obtained results are of vital importance for future studies on the expression of genes associated with the non-target-site resistance mechanisms in the A. spica-venti populations susceptible and resistant to herbicides.

scholarly journals Cloning and evaluation of reference genes for quantitative real-time PCR analysis inAmorphophallus

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3260 ◽  
Author(s):  
Kai Wang ◽  
Yi Niu ◽  
Qijun Wang ◽  
Haili Liu ◽  
Yi Jin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Real Time ◽  
Reference Genes ◽  
Dynamic Range ◽  
Expression Profiles ◽  
Mrna Levels ◽  
Expression Stability ◽  
Degenerate Primers ◽  
Different Tissues

Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes.Amorphophallusis a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes inAmorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genesAmorphophalluswere cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) inA. albusandA. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated thatEF1-a,EIF4A,H3andUBQwere the best reference genes under heat stress inAmorphophallus. Furthermore,EF1-a,EIF4A,TUB, andRPwere the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined thatEF1-α,EIF4A,andCYPwere stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock proteinSHSP, which is related to heat stress inAmorphophallus. In sum,EF1-αandEIF4Awere the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR inAmorphophallus.

scholarly journals Identification and Validation of Novel Reference Genes in Acute Lymphoblastic Leukemia for Droplet Digital PCR

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 376 ◽  
Author(s):  
Vanessa Villegas-Ruíz ◽  
Karina Olmos-Valdez ◽  
Kattia Alejandra Castro-López ◽  
Victoria Estefanía Saucedo-Tepanecatl ◽  
Josselen Carina Ramírez-Chiquito ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Reference Genes ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Housekeeping Genes ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cellular Processes

Droplet digital PCR is the most robust method for absolute nucleic acid quantification. However, RNA is a very versatile molecule and its abundance is tissue-dependent. RNA quantification is dependent on a reference control to estimate the abundance. Additionally, in cancer, many cellular processes are deregulated which consequently affects the gene expression profiles. In this work, we performed microarray data mining of different childhood cancers and healthy controls. We selected four genes that showed no gene expression variations (PSMB6, PGGT1B, UBQLN2 and UQCR2) and four classical reference genes (ACTB, GAPDH, RPL4 and RPS18). Gene expression was validated in 40 acute lymphoblastic leukemia samples by means of droplet digital PCR. We observed that PSMB6, PGGT1B, UBQLN2 and UQCR2 were expressed ~100 times less than ACTB, GAPDH, RPL4 and RPS18. However, we observed excellent correlations among the new reference genes (p < 0.0001). We propose that PSMB6, PGGT1B, UBQLN2 and UQCR2 are housekeeping genes with low expression in childhood cancer.

scholarly journals Sex bias evaluation of classic and novel Housekeeping Genes in adipose tissue through the massive analysis of transcriptomics data

2021 ◽  
Author(s):  
Maria Guaita-Cespedes ◽  
Rubén Grillo-Risco ◽  
Marta R. Hidalgo ◽  
Sonia Fernández-Veledo ◽  
Deborah Burks ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Expression Profiles ◽  
Housekeeping Genes ◽  
Sex Bias ◽  
Expression Stability ◽  
Expression Levels ◽  
Cell Functions ◽  
Web Resource ◽  
New Genes

ABSTRACTHousekeeping genes (HKG), those involved in the maintenance of basic cell functions, are considered to have constant expression levels in all cell types, and are therefore commonly used as internal controls in gene expression studies. Nevertheless, multiple studies have shown that not all of them have stable expression levels across different cells, tissues, and conditions, introducing a systematic error in the experimental results. The proper selection and validation of control housekeeping genes in the specific studied conditions is crucial for the validity of the obtained results, although, up to date, sex has never been taken into account as a biological variable.In this work, we evaluate the expression profiles of six classical housekeeping genes, (four metabolic: HPRT, GAPDH, PPIA and UBC, and two ribosomal: 18S and RPL19) used as controls in several tissues, to determine the stability of their expression in adipose tissue of Homo sapiens and Mus musculus and asses sex bias and control suitability. We also evaluated gene expression stability of the genes included in different whole transcriptome microarrays available at the Gene Expression Omnibus database (GEO), to identify new genes suitable to be used as sex-unbiased controls. We perform a sex-based analysis to test for/reveal sexual dimorphism of mRNA expression stability.We use a novel computational strategy based on meta-analysis techniques which evidence that some classical housekeeping genes do not fit to analyze human adipose tissue when sex variable is included. For instance, the extensively used 18S has shown to be variable in this tissue, while PPIA and RPL19 have shown to be good HKG targets. Further, we propose new sex-unbiased human and mouse housekeeping genes, derived from sex-specific expression profiles, including, RPS8 or UBB. All the results generated in this work are available in an open web resource (https://bioinfo.cipf.es/metafun-HKG), so that they can be consulted and used in further studies.

scholarly journals Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3137
Author(s):  
Juan Zhao ◽  
Cheng Wang ◽  
Lin Zhang ◽  
Aiai Lei ◽  
Linjie Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Target Genes ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Stability ◽  
Transport Pathway ◽  
Metabolic Role ◽  
Suitable Reference

As the largest chamber of the ruminant stomach, the rumen not only serves as the principal absorptive surface and nutrient transport pathway from the lumen into the animal, but also plays an important short-chain fatty acid (SCFA) metabolic role in addition to protective functions. Accurate characterization of the gene expression profiles of genes of interest is essential to the exploration of the intrinsic regulatory mechanisms of rumen development in goats. Thus, the selection of suitable reference genes (RGs) is an important prerequisite for real-time quantitative PCR (RT-qPCR). In the present study, 16 candidate RGs were identified from our previous transcriptome sequencing of caprine rumen tissues. The quantitative expressions of the candidate RGs were measured using the RT-qPCR method, and the expression stability of the RGs was assessed using the geNorm, NormFinder, and BestKeeper programs. GeNorm analysis showed that the M values were less than 0.5 for all the RGs except GAPT4, indicating that they were stably expressed in the rumen tissues throughout development. RPS4X and RPS6 were the two most stable RGs. Furthermore, the expressions of two randomly selected target genes (IGF1 and TOP2A), normalized by the selected most stable RGs (RPS4X and RPS6), were consistent with the results of RNA sequencing, while the use of GAPDH and ACTB as RGs resulted in altered profiles. Overall, RPS4X and RPS6 showed the highest expression stability and the lowest coefficients of variation, and could be used as the optimal reference combination for quantifying gene expression in rumen tissues via RT-qPCR analysis.

scholarly journals Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

2015 ◽  
Vol 2015 ◽  
pp. 1-20 ◽  
Author(s):  
Cora S. Thiel ◽  
Swantje Hauschild ◽  
Svantje Tauber ◽  
Katrin Paulsen ◽  
Christiane Raig ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Housekeeping Genes ◽  
Altered Gravity ◽  
Experimental Conditions ◽  
Expression Levels ◽  
Expression Studies ◽  
Gene Expression Studies

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

scholarly journals Identification of appropriate reference genes for gene expression studies in mice left ventricles from different developmental stages

2021 ◽  
Author(s):  
Jie Ren ◽  
Ningning Zhang ◽  
Xiangjie Li ◽  
Xiaogang Sun ◽  
Jiangping Song
Keyword(s):  
Gene Expression ◽  
Heart Development ◽  
Reference Genes ◽  
Developmental Stages ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Housekeeping Genes ◽  
Good Choice ◽  
Expression Stability ◽  
Gene Expressions

Abstract Aims: Real-time quantitative polymerase chain reaction (RT-qPCR) is the standard assay used for revealing the gene expression characteristics. However, the RT-qPCR studies all need reference genes for normalization to make the results comparable, which should hold a high expression stability during all experimental datasets. So far, there was no optimal set of reference genes identified in mice left ventricles (LV) across embryonic and postnatal stages. The objective of our research was to identify the appropriate reference genes in mice LV from different developmental stages.Methods and Results: we investigated the gene expressions of common 21 candidate housekeeping genes in mice LV from 7 different developmental stages, almost throughout the whole period of the mouse lifespan. The expression of some candidate reference genes, such as 18S and Actb, apparently fluctuated. The stability of potential reference genes was evaluated by a number of methods, such as GeNorm, NormFinder, BestKeeper, Delta-Ct and RefFinder method. we identified a set of optimal reference genes that can be reliably used for normalization of RT-qPCR experiments in different developmental stages of mice LV. Our results showed that following genes should not be used as reference genes in mice LV development studies: 18S, Hmbs, Ubc, Psmb4, Tfrc and Actb. And the Rplp0 appeared to represent a good choice. Conclusions: Our study provides the expression stability of the commonly used reference genes in process of LV development and maturation. We also identified a set of optimal reference genes under different conditions. Our findings may be helpful in future studies to investigate the gene expression patterns and mechanism of mammalian heart development.

scholarly journals Selection and Validation of Appropriate Reference Genes for Gene Expression Studies in Schima Superba

2021 ◽  
Author(s):  
Zhongyi Yang ◽  
Rui Zhang ◽  
Zhichun Zhou
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Geometric Mean ◽  
Gene Transcript ◽  
Expression Stability ◽  
Schima Superba ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Different Tissues

Abstract Background Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

scholarly journals Identification and validation of miRNA reference genes in poplar under pathogen stress

2021 ◽  
Author(s):  
Lichun Zhang ◽  
Xiaoqian Yang ◽  
Yiyi Yin ◽  
Jinxing Wang ◽  
Yanwei Wang
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Small Rna ◽  
Reference Genes ◽  
Expression Profiles ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Internal Standards ◽  
Qrt Pcr ◽  
Canker Pathogen

Abstract Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes especially miRNAs have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using three reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g and miR156a were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a was used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different lenth between detected miRNAs and traditional referece genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.

Functional Genomics Analysis of Horseweed (Conyza canadensis) with Special Reference to the Evolution of Non–Target-Site Glyphosate Resistance

Weed Science ◽  
2010 ◽  
Vol 58 (2) ◽  
pp. 109-117 ◽  
Author(s):  
Joshua S. Yuan ◽  
Laura L. G. Abercrombie ◽  
Yongwei Cao ◽  
Matthew D. Halfhill ◽  
Xin Zhou ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Molecular Mechanisms ◽  
Glyphosate Resistance ◽  
Resistance Mechanisms ◽  
Target Site ◽  
Environmentally Benign ◽  
Conyza Canadensis ◽  
Target Site Resistance ◽  
Weedy Species ◽  
Heterologous Microarray

The evolution of glyphosate resistance in weedy species places an environmentally benign herbicide in peril. The first report of a dicot plant with evolved glyphosate resistance was horseweed, which occurred in 2001. Since then, several species have evolved glyphosate resistance and genomic information about nontarget resistance mechanisms in any of them ranges from none to little. Here, we report a study combining iGentifier transcriptome analysis, cDNA sequencing, and a heterologous microarray analysis to explore potential molecular and transcriptomic mechanisms of nontarget glyphosate resistance of horseweed. The results indicate that similar molecular mechanisms might exist for nontarget herbicide resistance across multiple resistant plants from different locations, even though resistance among these resistant plants likely evolved independently and available evidence suggests resistance has evolved at least four separate times. In addition, both the microarray and sequence analyses identified non–target-site resistance candidate genes for follow-on functional genomics analysis.

Target and Non–target site Mechanisms Confer Resistance to Glyphosate in Canadian Accessions ofConyza canadensis

Weed Science ◽  
2017 ◽  
Vol 66 (2) ◽  
pp. 234-245 ◽  
Author(s):  
Eric R. Page ◽  
Christopher M. Grainger ◽  
Martin Laforest ◽  
Robert E. Nurse ◽  
Istvan Rajcan ◽  
...  
Keyword(s):  
Ssr Markers ◽  
Resistance Mechanisms ◽  
Target Site ◽  
Conyza Canadensis ◽  
Weed Species ◽  
Target Site Resistance ◽  
Selection For ◽  
Simple Sequence ◽  
Growing Region ◽  
Selection Of

Glyphosate-resistant populations ofConyza canadensishave been spreading at a rapid rate in Ontario, Canada, since first being documented in 2010. Determining the genetic relationship among existing Ontario populations is necessary to understand the spread and selection of the resistant biotypes. The objectives of this study were to: (1) characterize the genetic variation ofC. canadensisaccessions from the province of Ontario using simple sequence repeat (SSR) markers and (2) investigate the molecular mechanism (s) conferring resistance in these accessions. Ninety-eightC. canadensisaccessions were genotyped using 8 SSR markers. Germinable accessions were challenged with glyphosate to determine their dose response, and the sequences of 5-enolpyruvylshikimate-3-phosphate synthase genes 1 and 2 were obtained. Results indicate that a majority of glyphosate-resistant accessions from Ontario possessed a proline to serine substitution at position 106, which has previously been reported to confer glyphosate resistance in other crop and weed species. Accessions possessing this substitution demonstrated notably higher levels of resistance than non–target site resistant (NTSR) accessions from within or outside the growing region and were observed to form a subpopulation genetically distinct from geographically proximate glyphosate-susceptible and NTSR accessions. Although it is unclear whether other non–target site resistance mechanisms are contributing to the levels of resistance observed in target-site resistant accessions, these results indicate that, at a minimum, selection for Pro-106-Ser has occurred in addition to selection for non–target site resistance and has significantly enhanced the levels of resistance to glyphosate inC. canadensisaccessions from Ontario.

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