Identification and validation of suitable internal reference genes for SYBR-GREEN qRT-PCR studies during cucumber development

Abstract Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, β-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, β-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.

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Selection of reference genes for quantitative real-time PCR normalization in the coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae)

Bulletin of Entomological Research ◽

10.1017/s0007485321000596 ◽

2021 ◽

pp. 1-11

Author(s):

Qianqian Meng ◽

Benshui Shu ◽

Shiwei Sun ◽

Ying Wang ◽

Mei Yang ◽

...

Keyword(s):

Real Time ◽

Reference Genes ◽

Molecular Mechanisms ◽

Target Genes ◽

Stem Borer ◽

Internal Reference ◽

Experimental Conditions ◽

Tissue Samples ◽

Abiotic Conditions ◽

Qrt Pcr

Abstract The coffee white stem borer, Xylotrechus quadripes Chevrolat (Coleoptera: Cerambycidae), is a major destructive pest of Coffea arabica L. (Gentianales: Rubiaceae), widely planted in many Asian countries, including China. Quantitative real-time polymerase chain reaction (qRT-PCR) is a common method for quantitative analysis of gene transcription levels. To obtain accurate and reliable qRT-PCR results, it is necessary to select suitable reference genes to different experimental conditions for normalizing the target gene expression. However, the stability of the expression of reference genes in X. quadripes has rarely been studied. In this study, the expression stability of nine candidate reference genes were investigated under biotic and abiotic conditions for use in qRT-PCR's normalization. By integrating the results of four algorithms of NormFinder, BestKeeper, geNorm, and RefFinder, the optimal reference gene combinations in different experimental conditions were performed as follows: RPL10a and EIF3D were the optimal reference genes for developmental stage samples, EIF4E, RPL10a, and RPS27a for tissue samples, V-ATP and EF1α for the sex samples, EIF3D and V-ATP for temperature treatment, RPS27a and RPL10a for insecticide stress, and RPL10a, RPS27a, and EF1α for all the samples. This study will help to obtain the stable internal reference genes under biotic and abiotic conditions and lay the foundation for in-depth functional research of target genes or genomics on olfactory molecular mechanisms, temperature adaptability, and insecticide resistance in X. quadripes.

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Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes

Biomarker Insights ◽

10.4137/bmi.s5596 ◽

2010 ◽

Vol 5 ◽

pp. BMI.S5596 ◽

Cited By ~ 7

Author(s):

Yi-Hong Zhou ◽

Vinay R. Raj ◽

Eric Siegel ◽

Liping Yu

Keyword(s):

Gene Expression ◽

Real Time ◽

Gene Expression Data ◽

Reference Genes ◽

Pcr Analysis ◽

Expression Data ◽

Internal Reference ◽

Expression Ratio ◽

Qrt Pcr ◽

Single Standard

In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR) because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR) to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients–-a process that requires large sample sizes by combining independent sets of data.

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Screening and Evaluation of Stable Reference Genes for Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis in Chinese Fir Roots under Water, Phosphorus, and Nitrogen Stresses

Forests ◽

10.3390/f10121087 ◽

2019 ◽

Vol 10 (12) ◽

pp. 1087 ◽

Cited By ~ 2

Author(s):

Ranhong Chen ◽

Wanting Chen ◽

Mulualem Tigabu ◽

Weimin Zhong ◽

Yushan Li ◽

...

Keyword(s):

Gene Expression ◽

Drought Stress ◽

Reference Genes ◽

Chinese Fir ◽

Chain Reaction ◽

Internal Reference ◽

Qrt Pcr ◽

N Starvation ◽

Polymerase Chain ◽

The Stability

Chinese fir (Cunninghamia lanceolata) is an economical important timber species widely planted in southeastern Asia. Decline in yield and productivity during successive rotation is believed to be linked with abiotic stress, such as drought stress and nitrogen (N) and phosphorus (P) starvation. Molecular breeding could be an option to develop tolerant genotypes. For gene expression studies using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), stable reference genes are needed for normalization of gene expression under different experimental conditions. However, there is no internal reference genes identified for Chinese fir under abiotic stresses. Thus, nine internal reference genes based on transcriptome data were selected and analyzed in the root of Chinese fir under drought stress and N and P starvation. Data were analyzed using geNorm, NormFinder, and BestKeeper, to screen and identify the best reference genes. The results showed that the UBQ and GAPDH genes were the two most stable genes under drought stress and the Actin1 and GAPDH were the two most stable genes under P starvation. Further, it was discovered that the Actin1 and UBC were the two most stable genes under N starvation among nine candidate reference genes. The gene expression of drought stress induced expression protein 14-3-3-4, the P transporter gene ClPht1;3, and the nitrate transporter gene NRT1.1 were used to verify the stability of the selected reference genes under drought stress and P and N starvation, respectively, and the results revealed that the screened reference genes were sufficient to normalize expression of the target genes. In conclusion, the results demonstrate that the stability of reference genes was closely related to the external conditions and reference genes applied to the roots of Chinese fir under different abiotic stress treatments were different. Our data will facilitate further studies on stress ecology and gene function analysis in Chinese fir.

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Selection of Internal Reference Genes for Normalization of Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) Analysis in the Canine Brain and Other Organs

Molecular Biotechnology ◽

10.1007/s12033-012-9543-6 ◽

2012 ◽

Vol 54 (1) ◽

pp. 47-57 ◽

Cited By ~ 12

Author(s):

Sang-Je Park ◽

Jae-Won Huh ◽

Young-Hyun Kim ◽

Sang-Rae Lee ◽

Sang-Hyun Kim ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Reference Genes ◽

Pcr Analysis ◽

Chain Reaction ◽

Internal Reference ◽

Qrt Pcr ◽

Canine Brain ◽

Polymerase Chain ◽

Selection Of

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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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Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts

Physiological Genomics ◽

10.1152/physiolgenomics.00004.2012 ◽

2012 ◽

Vol 44 (12) ◽

pp. 651-656 ◽

Cited By ~ 6

Author(s):

S. Ellefsen ◽

M. Bliksøen ◽

A. Rutkovskiy ◽

I. B. Johansen ◽

M.-L. Kaljusto ◽

...

Keyword(s):

Infarct Size ◽

Real Time ◽

Reference Genes ◽

Stable Expression ◽

Unit Weight ◽

Living Tissue ◽

Rt Pcr ◽

Internal Reference ◽

Total Rna ◽

Rat Hearts

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability ( R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, β-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1–50.1%). When β-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.

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