Screening of optimal reference genes for qRT-PCR and preliminary exploration of cold resistance mechanisms in Prunus mume and Prunus sibirica varieties

Abstract Background: As an alpine plants，Rhododendron chrysanthum (R. chrysanthum) has evolved cold resistance mechanisms and become a valuable plant resource with the responsive mechanism of cold stress. Results: In my study, we adopt the phosphoproteomic and proteomic analysis combining with physiological measurement to illustrate the responsive mechanism of R. chrysanthum seedling under cold (4℃) stress. After chilling for 12 h, 350 significantly changed proteins and 274 significantly changed phosphoproteins were detected. COG analysis showed that significantly changed proteins and phosphoproteins were mainly involved in signal transduction and energy production and conversion under cold stress. The results indicated photosynthesis was inhibited under cold stress, but cold induced calcium-mediated signaling, reactive oxygen species (ROS) homeostasis and other transcription regulation factors could protect plants from destruction caused by cold stress. Conclusions: These data constitute a cold stress-responsive metabolic atlas in R. chrysanthum, which will springboard further investigations into the complex molecular mechanisms of plant cold adaptation.

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Selection of Suitable Reference Genes for Quantitative Real-time Polymerase Chain Reaction in Prunus mume during Flowering Stages and under Different Abiotic Stress Conditions

Journal of the American Society for Horticultural Science ◽

10.21273/jashs.139.2.113 ◽

2014 ◽

Vol 139 (2) ◽

pp. 113-122 ◽

Cited By ~ 21

Author(s):

Tao Wang ◽

Ruijie Hao ◽

Huitang Pan ◽

Tangren Cheng ◽

Qixiang Zhang

Keyword(s):

Polymerase Chain Reaction ◽

Abiotic Stress ◽

Real Time ◽

Reference Genes ◽

Late Embryogenesis Abundant Protein ◽

Prunus Mume ◽

Chain Reaction ◽

Qrt Pcr ◽

Polymerase Chain ◽

Suitable Reference

Mei (Prunus mume) is widely cultivated in eastern Asia owing to its favored ornamental characteristics and its tolerance for low temperatures. Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used method for gene expression analysis, requiring carefully selected reference genes to ensure data reliability. The aim of this study was to identify and evaluate reference genes for qRT-PCR in mei. Ten candidate reference genes were chosen, and their expression levels were assessed by qRT-PCR in four sample sets: 1) flowering mei; 2) mei undergoing abiotic stress; 3) different genotypes of Prunus species; and 4) all mei samples. The stability and suitability of the candidate reference genes were validated using commercially available software. We found that protein phosphatase 2A-1 (PP2A-1) and PP2A-2 were suitable reference genes for flowering with ubiquitin-conjugating enzyme E2 (UBC) also being suitable for different genotypes of Prunus species. UBC and actin (ACT) were most stably expressed under abiotic stress. Finally, the expression of an AGAMOUS homolog of Arabidopsis thaliana (PmAG) and a putative homolog of Group 2 late embryogenesis abundant protein gene in A. thaliana (PmLEA) were assessed to allow comparisons between selected candidate reference genes, highlighting the importance of careful reference gene selection.

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Evaluation of reference genes for miRNA expression analysis in Galeruca daurica (Coleoptera: Chrysomelidae) using qRT‐PCR

Entomological Research ◽

10.1111/1748-5967.12537 ◽

2021 ◽

Author(s):

Hai‐Chao Wang ◽

Ling Li ◽

Yan‐Yan Li ◽

Yao Tan ◽

Bao‐Ping Pang

Keyword(s):

Expression Analysis ◽

Mirna Expression ◽

Reference Genes ◽

Qrt Pcr

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Public-transcriptome-database-assisted selection and validation of reliable reference genes for qRT-PCR in rice

Science China Life Sciences ◽

10.1007/s11427-019-1553-5 ◽

2019 ◽

Vol 63 (1) ◽

pp. 92-101 ◽

Cited By ~ 2

Author(s):

Zhe Zhao ◽

Zixu Zhang ◽

Zhi Ding ◽

Hengjun Meng ◽

Rongxin Shen ◽

...

Keyword(s):

Reference Genes ◽

Qrt Pcr ◽

Transcriptome Database

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Choice of a Stable Set of Reference Genes for qRT-PCR Analysis in Amblyomma maculatum (Acari: Ixodidae)

Journal of Medical Entomology ◽

10.1603/me12123 ◽

2012 ◽

Vol 49 (6) ◽

pp. 1339-1346 ◽

Cited By ~ 15

Author(s):

Rebecca Browning ◽

Steven Adamson ◽

Shahid Karim

Keyword(s):

Reference Genes ◽

Stable Set ◽

Pcr Analysis ◽

Amblyomma Maculatum ◽

Qrt Pcr

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Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

Scientific Reports ◽

10.1038/s41598-021-92780-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Madhab Kumar Sen ◽

Kateřina Hamouzová ◽

Pavlina Košnarová ◽

Amit Roy ◽

Josef Soukup

Keyword(s):

Reference Gene ◽

Herbicide Resistance ◽

Reference Genes ◽

Gene Selection ◽

High Yield ◽

Suitable Reference Gene ◽

Grass Species ◽

Experimental Conditions ◽

Qrt Pcr ◽

Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

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A probe-based qRT-PCR method to profile immunological gene expression in blood of captive beluga whales (Delphinapterus leucas)

PeerJ ◽

10.7717/peerj.3840 ◽

2017 ◽

Vol 5 ◽

pp. e3840 ◽

Cited By ~ 5

Author(s):

Ming-An Tsai ◽

I-Hua Chen ◽

Jiann-Hsiung Wang ◽

Shih-Jen Chou ◽

Tsung-Hsien Li ◽

...

Keyword(s):

Gene Expression ◽

Immune System ◽

Reference Genes ◽

Animal Health ◽

Rna Extraction ◽

Disease Onset ◽

Delphinapterus Leucas ◽

Amplification Efficiency ◽

Beluga Whales ◽

Qrt Pcr

Cytokines are fundamental for a functioning immune system, and thus potentially serve as important indicators of animal health. Quantitation of mRNA using quantitative reverse transcription polymerase chain reaction (qRT-PCR) is an established immunological technique. It is particularly suitable for detecting the expression of proteins against which monoclonal antibodies are not available. In this study, we developed a probe-based quantitative gene expression assay for immunological assessment of captive beluga whales (Delphinapterus leucas) that is one of the most common cetacean species on display in aquariums worldwide. Six immunologically relevant genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis, and two validated housekeeping genes (PGK1 and RPL4) with stable expression were used as reference genes. Sixteen blood samples were obtained from four animals with different health conditions and stored in RNAlater™ solution. These samples were used for RNA extraction followed by qRT-PCR analysis. Analysis of gene transcripts was performed by relative quantitation using the comparative Cq method with the integration of amplification efficiency and two reference genes. The expression levels of each gene in the samples from clinically healthy animals were normally distributed. Transcript outliers for IL-2Rα, IL-4, IL-12, TNFα, and IFNγ were noticed in four samples collected from two clinically unhealthy animals. This assay has the potential to identify immune system deviation from normal state, which is caused by health problems. Furthermore, knowing the immune status of captive cetaceans could help both trainers and veterinarians in implementing preventive approaches prior to disease onset.

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

10.21203/rs.3.rs-225734/v1 ◽

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.

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