scholarly journals Reference gene selection for qRT-PCR assays inStellera chamaejasmesubjected to abiotic stresses and hormone treatments based on transcriptome datasets

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4535 ◽  
Author(s):  
Xin Liu ◽  
Huirui Guan ◽  
Min Song ◽  
Yanping Fu ◽  
Xiaomin Han ◽  
...  
Keyword(s):  
Gene Expression ◽  
Internal Control ◽  
Abiotic Stresses ◽  
Target Genes ◽  
Expression Patterns ◽  
Control Measures ◽  
Rapid Expansion ◽  
Pcr Analysis ◽  
Data Normalization ◽  
Qrt Pcr

BackgroundStellera chamaejasmeLinn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion ofS. chamaejasmehas greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization inS. chamaejasmehas been reported.MethodIn this study, 10 candidate RGs namely,18S,60S,CYP,GAPCP1,GAPDH2,EF1B,MDH,SAND,TUA1, andTUA6, were singled out from the transcriptome database ofS. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper.ResultOur results showed thatGAPCP1andEF1Bwere the best combination for the three abiotic stresses, whereasTUA6andSAND,TUA1andCYP,GAPDH2and60Swere the best choices for ABA, GA, and ETH treatment, respectively. Moreover,GAPCP1and60Swere assessed to be the best combination for all samples, and18Swas the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2andGI) further verified that the RGs that we selected were suitable for gene expression normalization.DiscussionThis work is the first attempt to comprehensively estimate the stability of RGs inS. chamaejasme. Our results provide suitable RGs for high-precision normalization in qRT-PCR analysis, thereby making it more convenient to analyze gene expression under these experimental conditions.

scholarly journals Selection and Validation of Reference Genes for the qRT-PCR Assays of Populus ussuriensis Gene Expression under Abiotic Stresses and Related ABA Treatment

Forests ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 476
Author(s):  
Ming Wei ◽  
Yingxi Chen ◽  
Mengqiu Zhang ◽  
Jingli Yang ◽  
Han Lu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cold Stress ◽  
Internal Control ◽  
Reference Genes ◽  
Target Genes ◽  
High Salinity ◽  
High Yield ◽  
Pcr Analysis ◽  
Qrt Pcr ◽  
Populus Ussuriensis

Populus ussuriensis Kom. is one of the most important tree species for forest renewal in the eastern mountainous areas of Northeast China due to its fast growth, high yield, and significant commercial and ecological value. The selection of optimal reference genes for the normalization of qRT-PCR data is essential for the analysis of relative gene expression. In this study, fourteen genes were selected and assessed for their expression stability during abiotic stress (drought, high salinity, and cold stress) and after the treatment with the drought-related hormone ABA. Three algorithms were used, geNorm, NormFinder, and BestKeeper, and a comprehensive ranking of candidate reference genes was produced based on their output. The most appropriate reference genes were UBQ10 and RPL24 for drought and ABA treatment, UBQ10 and TUB3 for cold stress, and UBQ10 and 60S rRNA for high salinity. Overall, UBQ10 was the most stable reference gene for use as an internal control, whereas PP2A was the least stable. The expression of two target genes (P5CS2 and GI) was used to further verify that the selected reference genes were suitable for gene expression normalization. This work comprehensively assesses the stability of reference genes in Populus ussuriensis and identifies suitable reference genes for normalization during qRT-PCR analysis.

scholarly journals Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data

2021 ◽  
Vol 22 (5) ◽  
pp. 2569
Author(s):  
Xue Bai ◽  
Tao Chen ◽  
Yuan Wu ◽  
Mingyong Tang ◽  
Zeng-Fu Xu
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Cyperus Esculentus ◽  
Transcriptome Data ◽  
Qrt Pcr ◽  
Below Ground ◽  
The Stability

Tiger nut (Cyperus esculentus), a perennial C4 plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

scholarly journals Reference Gene Selection for qRT-PCR Analyses of Luffa (Luffa cylindrica) Plants Under Abiotic Stress Conditions

2020 ◽  
Author(s):  
mindong chen ◽  
bin wang ◽  
yongping li ◽  
meijuan zeng ◽  
jianting liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Expression Patterns ◽  
Cold Treatment ◽  
Suitable Reference Gene ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Suitable Reference

Abstract Background: Quantitative real-time PCR (qRT-PCR) is one of the preferred methods for analyzing gene expression, and selecting suitable internal reference genes is an important prerequisite for the application of this technology. However, no systematic studies have been conducted on reference genes in luffa, resulting in limited investigations of luffa gene expression. Results: In this study, seven reference genes ( ACT , TUA , TUB , EF-1α , GAPDH , UBQ , and 18S ) were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H 2 O 2 , and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H 2 O 2 and drought treatments. In contrast, GAPDH was revealed as an unsuitable reference gene overall and for the heat, salt, H 2 O 2 , ABA, and drought treatments. Regarding the cold treatment, TUA was identified as an unsuitable reference gene. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase ( Cu/Zn-SOD ) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. Conclusions: The study data were used to compile a list of suitable reference genes for qRT-PCR analyses of the gene expression in luffa plants exposed to abiotic stresses. This work may provide the basis for future qRT-PCR-based investigations of the transcription of important functional genes in luffa.

scholarly journals Genetic Comparison of Stemness of Human Umbilical Cord and Dental Pulp

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Chung-Min Kang ◽  
Hyunok Kim ◽  
Je Seon Song ◽  
Byung-Jai Choi ◽  
Seong-Oh Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Umbilical Cord ◽  
Dental Pulp ◽  
Tooth Development ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Human Umbilical Cord ◽  
Tissue Samples ◽  
Qrt Pcr ◽  
Expression Of Genes

This study focuses on gene expression patterns and functions in human umbilical cord (UC) and dental pulp (DP) containing mesenchymal stem cells (MSCs). DP tissues were collected from 25 permanent premolars. UC tissue samples were obtained from three newborns. Comparative gene profiles were obtained using cDNA microarray analysis and the expression of tooth development-associated and MSC-related genes was assessed by the quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Genes related to cell proliferation, angiogenesis, and immune responses were expressed at higher levels in UC, whereas genes related to growth factor and receptor activity and signal transduction were more highly expressed in DP. Although UC and DP tissues exhibited similar expression of surface markers for MSCs, UC showed higher expression of CD29, CD34, CD44, CD73, CD105, CD146, and CD166. qRT-PCR analysis showed that CD146, CD166, and MYC were expressed 18.3, 8.24, and 1.63 times more highly in UC, whereas the expression of CD34 was 2.15 times higher in DP. Immunohistochemical staining revealed significant differences in the expression of genes (DSPP,DMP1, andCALB1) related to odontogenesis and angiogenesis in DP. DP and UC tissue showed similar gene expression, with the usual MSC markers, while they clearly diverged in their differentiation capacity.

scholarly journals Species diversity and spatial distribution of CL/VL vectors: assessing bioclimatic effect on expression plasticity of genes possessing vaccine properties isolated from wild-collected sand flies in endemic areas of Iran

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ali Bordbar ◽  
Parviz Parvizi
Keyword(s):  
Gene Expression ◽  
Spatial Distribution ◽  
Interpolation Method ◽  
Diversity Index ◽  
Fold Change ◽  
Control Measures ◽  
Pcr Analysis ◽  
Physiological Status ◽  
Sand Flies ◽  
Risk Maps

Abstract Background Leishmaniasis is one of the ten most important neglected tropical diseases worldwide. Understanding the distribution of vectors of visceral and cutaneous leishmaniasis (VL/CL) is one of the significant strategic frameworks to control leishmaniasis. In this study, the extent of the bioclimatic variability was investigated to recognize a rigorous cartographic of the spatial distribution of VL/CL vectors as risk-maps using ArcGIS modeling system. Moreover, the effect of bioclimatic diversity on the fold change expression of genes possessing vaccine traits (SP15 and LeIF) was evaluated in each bioclimatic region using real-time PCR analysis. Methods The Inverse Distance Weighting interpolation method was used to obtain accurate geography map in closely-related distances. Bioclimatic indices were computed and vectors spatial distribution was analyzed in ArcGIS10.3.1 system. Species biodiversity was calculated based on Shannon diversity index using Rv.3.5.3. Expression fold change of SP15 and LeIF genes was evaluated using cDNA synthesis and RT-qPCR analysis. Results Frequency of Phlebotomus papatasi was predominant in plains areas of Mountainous bioclimate covering the CL hot spots. Mediterranean region was recognized as an important bioclimate harboring prevalent patterns of VL vectors. Semi-arid bioclimate was identified as a major contributing factor to up-regulate salivary-SP15 gene expression (P = 0.0050, P < 0.05). Also, Mediterranean bioclimate had considerable effect on up-regulation of Leishmania-LeIF gene in gravid and semi-gravid P. papatasi population (P = 0.0109, P < 0.05). Conclusions The diversity and spatial distribution of CL/VL vectors associated with bioclimatic regionalization obtained in our research provide epidemiological risk maps and establish more effectively control measures against leishmaniasis. Oscillations in gene expression indicate that each gene has its own features, which are profoundly affected by bioclimatic characteristics and physiological status of sand flies. Given the efficacy of species-specific antigens for vaccine production, it is essential to consider bioclimatic factors that have a fundamental role in affecting the regulatory regions of environmentally responsive loci for genes used in vaccine design.

scholarly journals GENECFE-ANFIS: A NEURO-FUZZY INFERENCE SYSTEM TO INFER GENE-GENE INTERACTIONS BASED ON RECOGNITION OF MICROARRAY GENE EXPRESSION PATTERNS

2007 ◽  
Vol 19 (02) ◽  
pp. 71-78 ◽  
Author(s):  
Cheng-Long Chuang ◽  
Chung-Ming Chen ◽  
Grace S. Shieh ◽  
Joe-Air Jiang
Keyword(s):  
Gene Expression ◽  
Fuzzy Inference System ◽  
Fuzzy Inference ◽  
Expression Patterns ◽  
Gene Interactions ◽  
Microarray Gene Expression ◽  
Inference System ◽  
Qrt Pcr ◽  
Neuro Fuzzy ◽  
Microarray Gene

A neuro-fuzzy inference system that recognizes the expression patterns of genes in microarray gene expression (MGE) data, called GeneCFE-ANFIS, is proposed to infer gene interactions. In this study, three primary features are utilized to extract genes' expression patterns and used as inputs to the neuro-fuzzy inference system. The proposed algorithm learns expression patterns from the known genetic interactions, such as the interactions confirmed by qRT-PCR experiments or collected through text-mining technique by surveying previously published literatures, and then predicts other gene interactions according to the learned patterns. The proposed neuro-fuzzy inference system was applied to a public yeast MGE dataset. Two simulations were conducted and checked against 112 pairs of qRT-PCR confirmed gene interactions and 77 TFs (Transcriptional Factors) pairs collected from literature respectively to evaluate the performance of the proposed algorithm.

Discovery and Characterization of Differentially Expressed Soybean MiRNAs and Their Targets During Soybean Mosaic Virus Infection Unveils Novel Insight Into Soybean-SMV Interaction

2021 ◽  
Author(s):  
Bowen Li ◽  
Adhimoolam Karthikeyan ◽  
Liqun Wang ◽  
Jinlong Yin ◽  
Tongtong Jin ◽  
...  
Keyword(s):  
Virus Infection ◽  
Mosaic Virus ◽  
Target Genes ◽  
Soybean Mosaic Virus ◽  
Expression Patterns ◽  
Defense Responses ◽  
Pcr Analysis ◽  
Functional Annotations ◽  
Additional Information ◽  
Insight Into

Abstract Background: Soybean mosaic virus (SMV) is the most devastating pathogen of soybean. MicroRNAs (miRNAs) are a class of non-coding RNAs (21-24 nucleotides) and play important roles in regulating defense responses against pathogens. However, miRNA's response to SMV in soybean is not as well documented. Result: In this study, we analyzed 18 miRNA libraries, including three biological replicates from two soybean lines (Resistant and susceptible lines to SMV strain SC3 selected from the near-isogenic lines of Qihuang No. 1× Nannong1138-2) after virus infection at three different time intervals (0 dpi, 7 dpi, and 14 dpi). A total of 1,092 miRNAs, including 608 known miRNAs and 484 novel miRNAs were detected. Differential expression analyses identified the miRNAs responded during soybean-SMV interaction. Then, miRNAs potential target genes were predicted via data mining, and functional annotation was done by Gene Ontology (GO) analysis. Eventually, the expression patterns of several miRNAs validated by quantitative real-time PCR analysis are consistent with sequencing results. Conclusion: We have identified a large number of miRNAs and their target genes and also functional annotations. Our study provides additional information on soybean miRNAs and an insight into the role of miRNAs during SMV-infection in soybean.

scholarly journals Genome-Wide Identification and Expression Profiling Analysis of the Galactinol Synthase Gene Family in Cassava (Manihot esculenta Crantz)

Agronomy ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 250 ◽  
Author(s):  
Ruimei Li ◽  
Shuai Yuan ◽  
Yingdui He ◽  
Jie Fan ◽  
Yangjiao Zhou ◽  
...  
Keyword(s):  
Gene Family ◽  
Abiotic Stresses ◽  
Manihot Esculenta ◽  
Expression Patterns ◽  
Pcr Analysis ◽  
Systematic Research ◽  
Raffinose Family Oligosaccharides ◽  
Manihot Esculenta Crantz ◽  
Galactinol Synthase ◽  
Genome Wide

Galactinol synthases (GolSs) are the key enzymes that participate in raffinose family oligosaccharides (RFO) biosynthesis, which perform a big role in modulating plant growth and response to biotic or abiotic stresses. To date, no systematic study of this gene family has been conducted in cassava (Manihot esculenta Crantz). Here, eight MeGolS genes are isolated from the cassava genome. Based on phylogenetic background, the MeGolSs are clustered into four groups. Through predicting the cis-elements in their promoters, it was discovered that all MeGolS members act as hormone-, stress-, and tissue-specific related elements to different degrees. MeGolS genes exhibit incongruous expression patterns in various tissues, indicating that different MeGolS proteins might have diverse functions. MeGolS1 and MeGolS3–6 are highly expressed in leaves and midveins. MeGolS3–6 are highly expressed in fibrous roots. Quantitative real-time Polymerase Chain Reaction (qRT-PCR) analysis indicates that several MeGolSs, including MeGolS1, 2, 5, 6, and 7, are induced by abiotic stresses. microRNA prediction analysis indicates that several abiotic stress-related miRNAs target the MeGolS genes, such as mes-miR156, 159, and 169, which also respond to abiotic stresses. The current study is the first systematic research of GolS genes in cassava, and the results of this study provide a basis for further exploration the functional mechanism of GolS genes in cassava.

scholarly journals Key Regulatory Pathways, MicroRNAs, and Target Genes Participate in Adventitious Root Formation of Acer Rubrum L

2021 ◽  
Author(s):  
Wenpeng Zhu ◽  
Manyu Zhang ◽  
Jianyi Li ◽  
Hewen Zhao ◽  
Kezhong Zhang ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Seedling Survival ◽  
Expression Patterns ◽  
Economic Value ◽  
Acer Rubrum ◽  
Pcr Analysis ◽  
Asexual Propagation ◽  
Regulatory Pathways

Abstract BackgroundAcer rubrum L. is a colorful ornamental tree with great economic value. Because this tree is difficult to root under natural conditions and the seedling survival rate is low, vegetative propagation methods are often used. Because the formation of adventitious roots (ARs) is essential for the survival of asexual propagation of A. rubrum, it is necessary to investigate the molecular regulatory mechanisms in the formation of ARs of A. ruburm. To address this knowledge gap, we sequenced the transcriptome and sRNA of the A. rubrum variety ‘Autumn Fantasy’ using high-throughput sequencing and explored changes in gene and microRNA (miRNA) expression in response to exogenous auxin treatment. ResultsWe identified 82,468 differentially expressed genes between the treated and untreated ARs, as well as 48 known and 95 novel miRNAs. We also identified 172 target genes of the known miRNAs using degradome sequencing. Two regulatory pathways (ubiquitin mediated proteolysis and plant hormone signal transduction), Ar-miR160a and the target gene ArARF10 were shown to be involved in the auxin response. We further investigated the expression patterns and regulatory roles of ArARF10 through subcellular localization, transcriptional activation, plant transformation, qRT-PCR analysis, and GUS staining. ConclusionsDifferential expression patterns indicated the Ar-miR160a-ArARF10 interaction might play a significant role in the regulation of AR formation in A. rubrum. Our study provided new insights into mechanisms underlying the regulation of AR formation in A. rubrum.

scholarly journals Alterations of Growth and Focal Adhesion Molecules in Human Breast Cancer Cells Exposed to the Random Positioning Machine

2021 ◽  
Vol 9 ◽  
Author(s):  
Jayashree Sahana ◽  
Thomas J. Corydon ◽  
Markus Wehland ◽  
Marcus Krüger ◽  
Sascha Kopp ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Gene Expression ◽  
Target Genes ◽  
Interaction Analysis ◽  
Expression Patterns ◽  
Focal Adhesions ◽  
Multicellular Spheroids ◽  
Down Regulation ◽  
Random Positioning Machine ◽  
Mcf 7

In this study, we evaluated changes in focal adhesions (FAs) in two types of breast cancer cell (BCC) lines (differentiated MCF-7 and the triple-negative MDA-MB-231 cell line) exposed to simulated microgravity (s-μg) created by a random positioning machine (RPM) for 24 h. After exposure, the BCC changed their growth behavior and exhibited two phenotypes in RPM samples: one portion of the cells grew as a normal two-dimensional monolayer [adherent (AD) BCC], while the other portion formed three-dimensional (3D) multicellular spheroids (MCS). After 1 h and 30 min (MDA-MB-231) and 1 h 40 min (MCF-7), the MCS adhered completely to the slide flask bottom. After 2 h, MDA-MB-231 MCS cells started to migrate, and after 6 h, a large number of the cells had left the MCS and continued to grow in a scattered pattern, whereas MCF-7 cells were growing as a confluent monolayer after 6 h and 24 h. We investigated the genes associated with the cytoskeleton, the extracellular matrix and FAs. ACTB, TUBB, FN1, FAK1, and PXN gene expression patterns were not significantly changed in MDA-MB-231 cells, but we observed a down-regulation of LAMA3, ITGB1 mRNAs in AD cells and of ITGB1, TLN1 and VCL mRNAs in MDA-MB-231 MCS. RPM-exposed MCF-7 cells revealed a down-regulation in the gene expression of FAK1, PXN, TLN1, VCL and CDH1 in AD cells and PXN, TLN and CDH1 in MCS. An interaction analysis of the examined genes involved in 3D growth and adhesion indicated a central role of fibronectin, vinculin, and E-cadherin. Live cell imaging of eGFP-vinculin in MCF-7 cells confirmed these findings. β-catenin-transfected MCF-7 cells revealed a nuclear expression in 1g and RPM-AD cells. The target genes BCL9, MYC and JUN of the Wnt/β-catenin signaling pathway were differentially expressed in RPM-exposed MCF-7 cells. These findings suggest that vinculin and β-catenin are key mediators of BCC to form MCS during 24 h of RPM-exposure.

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