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

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.

Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

2016 ◽  
Vol 107 (3) ◽  
pp. 359-368 ◽  
Author(s):  
Y. Tan ◽  
X.-R. Zhou ◽  
B.-P. Pang
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Target Genes ◽  
Gene Selection ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Functional Studies ◽  
Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔCt method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

scholarly journals Screening Potential Reference Genes in Tuta absoluta with Real-Time Quantitative PCR Analysis under Different Experimental Conditions

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1253
Author(s):  
An-Pei Yang ◽  
Yu-Sheng Wang ◽  
Cong Huang ◽  
Zhi-Chuang Lv ◽  
Wan-Xue Liu ◽  
...  
Keyword(s):  
Reference Genes ◽  
Molecular Mechanisms ◽  
Developmental Stages ◽  
Tuta Absoluta ◽  
Pcr Analysis ◽  
Internal Reference ◽  
Experimental Conditions ◽  
Real Time Quantitative Pcr ◽  
Adult Tissues ◽  
Induced Stresses

Tuta absoluta is one of the most significant invasive pests affecting tomato plants worldwide. RT-qPCR has emerged as one of the most sensitive and accurate methods for detecting gene expression data. The screening of stable internal reference genes is the most critical step for studying the molecular mechanisms of environmental adaptability. The stable reference genes expressed in T. absoluta under specific experimental conditions have not yet been clarified. In this study, seven candidate reference genes (RPL27, RPS13, RPS15, EF1-α, TUB, TBP, and β-actin) and their optimal numbers were evaluated under biotic (developmental stages and adult tissues) and abiotic (insecticide, temperature, and plant VOC) conditions using four software programs. Our results identified the following reference genes and numbers as optimal: three genes (EF1-α, RPS13, and RPL27) for different developmental stages (egg, larva, pupa, unmated adult), two genes (RPS13 and TBP) for adult tissues (antenna, head, thorax, abdomen, leg), two genes (TBP and RPS13) for insecticides (Bacillus thuringiensis, chlorpyrifos, abamectin-aminomethyl, and chlorantraniliprole), two genes (RPL27 and TUB) for temperature-induced stresses (0, 25, and 40 °C), and two genes (RPS13 and TUB) for VOC-induced stresses (nonanal, α-phellandrene, and tomato leaves). Our results provide a reference for selecting appropriate reference genes for further study of the functional genes of T. absoluta under different experimental conditions.

scholarly journals Identification and Evaluation of Reference Genes for Normalization of Gene Expression in Developmental Stages, Sexes, and Tissues of Diaphania caesalis (Lepidoptera, Pyralidae)

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Zheng Wang ◽  
Qianqian Meng ◽  
Xi Zhu ◽  
Shiwei Sun ◽  
Aiqin Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Target Gene ◽  
Target Genes ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Transcriptional Level ◽  
Internal Reference ◽  
Target Gene Expression ◽  
Qrt Pcr

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.

Selection and validation of reference genes for quantitative real-time PCR analysis of Nitraria tangutorum

2020 ◽  
Author(s):  
Bo Wang ◽  
Lirong WANG ◽  
Huirong Duan ◽  
Peifang Chong ◽  
Shiping Su ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Expression Stability ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Nitraria Tangutorum ◽  
Desert Areas ◽  
Polymerase Chain ◽  
Suitable Reference

Abstract Background: Suitable reference genes can be used to calibrate the error in quantitative real‑time polymerase chain reaction (qRT-PCR) experiments and make the results more credible. However, reference genes suitable for different species and different experimental conditions do not exist. Nitraria tangutorum Bobr. is a typical plant in desert areas and desert plains, which is drought-resistant, saline-alkali resistant, barren-resistant, and has extremely strong adaptability. Due to insufficient understanding of the importance of this germplasm in the past, it is still unclear which genes can be used as reference genes to calibrate qRT-PCR data of N. tangutorum .Results: In this study, we analyzed the expression levels of 10 candidate reference genes (ACT, GAPDH, TUA, TUB, CYP, UBC, His, PP2A, HSP, and EF1-α) in three tissues (root, stem and leaf) and under five abiotic stresses (salt, drought, heat, cold, and ABA) of N. tangutorum seedlings by qRT-PCR. Three analysis software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate expression stability of ten genes. Comprehensive analysis showed that EF1-α and His had the best expression stability, whereas HSP was the least suitable as a reference gene. The expression profile of NtCER7, a gene related to the regulation of the waxy synthesis of N. tangutorum, verified the accuracy of the experimental results.Conclusion: Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This study provides the first data on stable reference genes in N. tangutorum, which will be beneficial to study of the gene expression of N. tangutorum and other Nitraria species in the future.

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

2010 ◽  
Vol 5 ◽  
pp. BMI.S5596 ◽  
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.

scholarly journals Identification and Validation of Reference Genes for RT-qPCR Analysis in Switchgrass under Heavy Metal Stresses

Genes ◽  
2020 ◽  
Vol 11 (5) ◽  
pp. 502 ◽  
Author(s):  
Junming Zhao ◽  
Man Zhou ◽  
Yu Meng
Keyword(s):  
Heavy Metal ◽  
Reference Gene ◽  
Reference Genes ◽  
18S Rrna ◽  
Target Genes ◽  
Heavy Metal Contamination ◽  
Internal Reference ◽  
Experimental Conditions ◽  
Leaves And Roots ◽  
Root Tissues

Switchgrass (Panicum Virgatum L.) has been recognized as the new energy plant, which makes it ideal for the development of phytoremediation on heavy metal contamination in soils with great potential. This study aimed to screen the best internal reference genes for the real-time quantitative PCR (RT-qPCR) in leaves and roots of switchgrass for investigating its response to various heavy metals, such as cadmium (Cd), lead (Pb), mercury (Hg), chromium (Cr), and arsenic (As). The stability of fourteen candidate reference genes was evaluated by BestKeeper, GeNorm, NormFinder, and RefFinder software. Our results identified U2AF as the best reference gene in Cd, Hg, Cr, and As treated leaves as well as in Hg, Pb, As, and Cr stressed root tissues. In Pb treated leaf tissues, 18S rRNA was demonstrated to be the best reference gene. CYP5 was determined to be the optimal reference gene in Cd treated root tissues. The least stable reference gene was identified to be CYP2 in all tested samples except for root tissues stressed by Pb. To further validate the initial screening results, we used the different sets of combinatory internal reference genes to analyze the expression of two metal transport associated genes (PvZIP4 and PvPDB8) in young leaves and roots of switchgrass. Our results demonstrated that the relative expression of the target genes consistently changed during the treatment when CYP5/UBQ1, U2AF/ACT12, eEF1a/U2AF, or 18S rRNA/ACT12 were combined as the internal reference genes. However, the time-dependent change pattern of the target genes was significantly altered when CYP2 was used as the internal reference gene. Therefore, the selection of the internal reference genes appropriate for specific experimental conditions is critical to ensure the accuracy and reliability of RT-qPCR. Our findings established a solid foundation to further study the gene regulatory network of switchgrass in response to heavy metal stress.

geNorm assessment of the efficacy of novel reference genes ALG9, TFCP2 and ZNF410 compared to GAPDH for real time normalization in GI carcinoids

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20052-20052
Author(s):  
G. Eick ◽  
M. Kidd ◽  
S. Mane ◽  
B. Nadler ◽  
M. Champaneria ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Large Scale ◽  
Target Genes ◽  
Marker Genes ◽  
Stable Gene ◽  
Rt Pcr ◽  
Experimental Conditions ◽  
Expression Levels ◽  
Accurate Normalization

20052 Background: Robust quantitation of potential clinical marker genes using quantitative real-time PCR (Q RT-PCR) is critically dependent on accurate normalization. Although GAPDH has historically been used for normalization, its expression has been shown to vary widely between different tissues and experimental conditions. Additionally, conventional normalization strategies based on a single housekeeping gene can lead to large normalization errors. The determination of a panel of genes that have robust expression in the experimental system being studied is therefore essential to ensure accurate normalization and interpretation of results. Methods: Based upon the availability of large-scale gene databases, we developed methodology to identify highly expressed genes (mean log-transformed expression levels: 4–8 in all samples) with low variability (S.D. < 0.22) in a Affymetrix U133A dataset of 36 gastrointestinal tumors and normal tissues. Eight novel candidate reference genes were identified and their expression levels established by Q RT-PCR in an independent set of GI tissue samples (n = 24). The geNorm tool was used to identify the most stably expressed set of genes amongst the 8 candidate genes. The expression levels of 3 potential GI tumor marker genes, namely the adhesin MAGE-D2, the metastasis-associated MTA1, and the mitosis regulator, NAP1L1, were normalized to GAPDH or geNorm and compared. Results: geNorm identified 3 genes, ALG9, TFCP2 and ZNF410, as the most robustly expressed control genes. GAPDH, in contrast, exhibited the highest variability and was considered the least stable gene of the nine evaluated. Two previously-identified target genes, MAGE-D2 and MTA1, were significantly elevated (p < 0.05) in malignant tumor samples (vs normal GI samples) when normalized by geNormATZ but not when normalized using GAPDH. NAP1L1 was only over-expressed in small intestinal carcinoids (normalized to geNormATZ). Expression levels of this gene were high in normal gastric mucosa. Conclusions: We provide a robust basis for the establishment of a reference gene set using GeneChip data and provide evidence for the clinical utility of identifying reference genes using the geNorm approach in GI neuroendocrine tumors. No significant financial relationships to disclose.

scholarly journals Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover (Trifolium repens L.) Involved in Five Abiotic Stresses

Plants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 996
Author(s):  
Qi Pu ◽  
Zhou Li ◽  
Gang Nie ◽  
Jiqiong Zhou ◽  
Lin Liu ◽  
...  
Keyword(s):  
Real Time ◽  
White Clover ◽  
Trifolium Repens ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Grass Species ◽  
Stable Gene ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Trifolium Repens L

White clover (Trifolium repens L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC, and ABC) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that 60SrRNA and ACT101 were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, SAMDC, UBQ, 60SrRNA, and ACT101 were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of Cyt-Cu/Zn SOD and CAT confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.

scholarly journals Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis of Development and Tissue-Dependent Flower Color Formation in Cymbidium lowianum

2022 ◽  
Vol 23 (2) ◽  
pp. 738
Author(s):  
Xiu-Mei Dong ◽  
Wei Zhang ◽  
Shi-Bao Zhang
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Molecular Mechanisms ◽  
Gene Selection ◽  
Expression Profiles ◽  
Flower Color ◽  
Pcr Analysis ◽  
Color Formation ◽  
Qrt Pcr

The development and tissue-dependent color formation of the horticultural plant results in various color pattern flowers. Anthocyanins and carotenoids contribute to the red and yellow colors, respectively. In this study, quantitative real-time polymerase chain reaction (qRT-PCR) is used to analyze the expression profiles of anthocyanin and carotenoids biosynthesis genes in Cymbidium lowianum (Rchb.f.) Rchb.f. Appropriate reference gene selection and validation are required before normalization of gene expression in qRT-PCR analysis. Thus, we firstly selected 12 candidate reference genes from transcriptome data, and used geNorm and Normfinder to evaluate their expression stability in lip (divided into abaxial and adaxial), petal, and sepal of the bud and flower of C. lowianum. Our results show that the two most stable reference genes in different tissues of C. lowianum bud and flower are EF1δ and 60S, the most unstable reference gene is 26S. The expression profiles of the CHS and BCH genes were similar to FPKM value profiles after normalization to the two most stable reference genes, EF1δ and 60S, with the upregulated CHS and BCH expression in flower stage, indicating that the ABP and CBP were activated across the stages of flower development. However, when the most unstable reference gene, 26S, was used to normalize the qRT-PCR data, the expression profiles of CHS and BCH differed from FPKM value profiles, indicating the necessity of selecting stable reference genes. Moreover, CHS and BCH expression was highest in the abaxial lip and adaxial lip, respectively, indicating that the ABP and CBP were activated in abaxial and adaxial lip, respectively, resulting in a presence of red or yellow segments in abaxial and adaxial lip. This study is the first to provide reference genes in C. lowianum, and also provide useful information for studies that aim to understand the molecular mechanisms of flower color formation in C. lowianum.

scholarly journals Optimizations for identifying reference genes in bone and cartilage bioengineering

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fei Xiong ◽  
Xiangyun Cheng ◽  
Chao Zhang ◽  
Roland Manfred Klar ◽  
Tao He
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Real Time ◽  
Reference Gene ◽  
Muscle Tissue ◽  
Reference Genes ◽  
Target Genes ◽  
The Stability ◽  
And Cartilage

Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

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