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 Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Bo Wang ◽  
Huirong Duan ◽  
Peifang Chong ◽  
Shiping Su ◽  
Lishan Shan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Expression Stability ◽  
Quantitative Real Time Pcr ◽  
Experimental Conditions ◽  
Nitraria Tangutorum ◽  
Multiple Species ◽  
Suitable Reference

Abstract Suitable reference genes can be used to calibrate the error in quantitative real-time PCR (qPCR) experiments, making the results more credible. However, there are no reference genes suitable for multiple species and under different experimental conditions. Nitraria tangutorum Bobr. is a typical plant native to desert areas. It is drought-resistant, saline-alkali resistant, extreme temperatures-resistant, and has strong adaptability. To date, the importance of this germplasm has not been sufficiently understood; therefore, it is still unclear which genes can be used as reference genes to calibrate qPCR data of N. tangutorum. 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 N. tangutorum seedlings under a series of experimental conditions, including in different organs (root, stem, and leaf) and under abiotic stresses (salt, drought, heat, and cold) and hormone stimuli (abscisic acid) by qPCR. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of the 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 cuticular wax biosynthesis in N. tangutorum, verified the accuracy of the experimental results. Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This paper provides the first data on stable reference genes in N. tangutorum, which will be beneficial to studying the gene expression of N. tangutorum and other Nitraria species in the future.

scholarly journals Identification And Evaluation of Reference Genes For Cynanchum Auriculatum Under Various Stress Conditions

2022 ◽  
Author(s):  
Zhi-Peng Zhu ◽  
Jian-Xiang Yu ◽  
Ke-Xin Wu ◽  
Qin-Yi Xu ◽  
Yi-Jun Kang ◽  
...  
Keyword(s):  
Reference Genes ◽  
Herbal Medicines ◽  
Stress Conditions ◽  
Pcr Analysis ◽  
Expression Stability ◽  
Qrt Pcr ◽  
Chinese Herbal ◽  
Cynanchum Auriculatum ◽  
Screening Study ◽  
Suitable Reference

Abstract Baishouwu (Cynanchum auriculatum) is a kind of critical Chinese herbal medicine. However, compared with the studies of other Chinese herbal medicines, the screening study on the reference genes of C. auriculatum is still the blank. Deterioration of the natural environment severely affects the growth and development of C. auriculatum. This study screened and identified suitable reference genes of C. auriculatum under various stress conditions. Based on qRT-PCR, geNorm, NormFinder, BestKeeper, and RefFinder were used for the expression stability evaluation of 12 potential reference genes from C. auriculatum. The ranking table showed that optimal reference genes included EF2 and SAMDC (heat stress), CYP and TUB-β (cold stress), TUB-α and GAPDH (drought stress), SAMDC and TUB-α (waterlogging stress), along with EF2 and ACT7 (salt stress). These results also demonstrated that under different abiotic stresses, suitable reference genes of plants should be selected for qRT-PCR analysis.

scholarly journals Evaluation of suitable reference genes for normalization of quantitative real-time PCR analysis in rice plants under Xanthomonas oryzae pv. oryzae--infection and melatonin supplementation

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
Xian Chen ◽  
Pedro Laborda ◽  
Yan Dong ◽  
Fengquan Liu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Housekeeping Genes ◽  
Suitable Reference Gene ◽  
Pcr Analysis ◽  
Quantitative Real Time Pcr ◽  
Rice Plants ◽  
Qrt Pcr ◽  
Suitable Reference

Abstract Exogenous melatonin (MT) was found to be an interesting tool for enhancing the resistance of rice to Xanthomonasoryzaepv. oryzae (Xoo)-caused bacterial blight (BB). However, the accurate comparison of the expression levels across samples was a challenging task. In this work, the stability of 10 common used housekeeping genes under Xoo-infection and MT supplementation in rice was analyzed using quantitative real-time PCR (qRT-PCR), and algorithms geNorm, NormFinder and BestKeeper. Our results indicated that most reference genes remained stable in Xoo-infected rice plants, while a number of reference genes were affected by MT supplementation. Among all studied genes, the transcript levels of 18S(18S ribosomal RNA) and UBC (Ubiquitin-conjugating enzyme E2) remained unaltered by Xoo infection, while UBC and UBQ5(Ubiquitin 5) were the most stable genes when examining simultaneous Xoo-infection and MT supplementation, demonstrating that UBC is a suitable reference gene for qRT-PCR data normalization in rice under Xoo-infection and MT supplementation.

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

2014 ◽  
Vol 139 (2) ◽  
pp. 113-122 ◽  
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.

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

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.

scholarly journals Selection and Validation of Reference Genes for qRT-PCR in Lentinula edodes under Different Experimental Conditions

Genes ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 647 ◽  
Author(s):  
Yi Luo ◽  
Gangzheng Wang ◽  
Chen Wang ◽  
Yuhua Gong ◽  
Yinbing Bian ◽  
...  
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Lentinula Edodes ◽  
Polymerase Chain Reaction Analysis ◽  
Pairwise Variation ◽  
Accurate Estimation ◽  
Expression Stability ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Accurate Normalization

Lentinula edodes is the most consumed mushroom in Asia due to its nutritional and medicinal values, and the optimal reference gene is crucial for normalization of its gene expression analysis. Here, the expression stability of 18 candidate reference genes (CRGs) in L. edodes was analyzed by three statistical algorithms (geNorm, NormFinder and BestKeeper) under different stresses (heat, cadmium excess and Trichoderma atroviride infection), different substrates (straw, sawdust and corn stalk) and different development stages (mycelia, primordia and fruit bodies). Among the 18 CRGs, 28S, Actin and α-tub exhibited the highest expression stability in L. edodes under all conditions, while GPD, SPRYP and MSF showed the least stable expression. The best reference gene in different conditions was different. The pairwise variation values showed that two genes would be sufficient for accurate normalization under different conditions of L. edodes. This study will contribute to more accurate estimation of the gene relative expression levels under different conditions using the optimal reference gene in qRT-PCR (quantitative reverse transcription polymerase chain reaction) analysis.

scholarly journals Comparison of Reliable Reference Genes Following Different Hormone Treatments by Various Algorithms for qRT-PCR Analysis of Metasequoia

2018 ◽  
Vol 20 (1) ◽  
pp. 34 ◽  
Author(s):  
Jing-Jing Wang ◽  
Shuo Han ◽  
Weilun Yin ◽  
Xinli Xia ◽  
Chao Liu
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Elongation Factor ◽  
Pcr Analysis ◽  
Qrt Pcr ◽  
Gene Normalization ◽  
Accurate Normalization ◽  
Suitable Reference ◽  
Different Tissues ◽  
Gene Expression Levels

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60β (60-kDa chaperonin β-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.

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 Evaluation of potential reference genes for quantitative RT-PCR analysis in spotted sea bass (Lateolabrax maculatus) under normal and salinity stress conditions

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5631 ◽  
Author(s):  
Haolong Wang ◽  
Haishen Wen ◽  
Yun Li ◽  
Kaiqiang Zhang ◽  
Yang Liu
Keyword(s):  
Salinity Stress ◽  
Reference Genes ◽  
18S Rrna ◽  
Sea Bass ◽  
Stress Conditions ◽  
Pcr Analysis ◽  
Stable Gene ◽  
Qrt Pcr ◽  
Lateolabrax Maculatus ◽  
Suitable Reference

The aim of this study was to select the most suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) of spotted sea bass (Lateolabrax maculatus), an important commercial marine fish in Pacific Asia, under normal physiological and salinity stress conditions. A total of 9 candidate reference genes (HPRT, GAPDH, EF1A, TUBA, RPL7, RNAPol II, B2M, ACTB and 18S rRNA) were analyzed by qRT-PCR in 10 tissues (intestine, muscle, stomach, brain, heart, liver, gill, kidney, pectoral fins and spleen) of L. maculatus. Four algorithms, geNorm, NormFinder, BestKeeper, and comparative ΔCt method, were used to evaluate the expression stability of the candidate reference genes. The results showed the 18S rRNA was most stable in different tissues under normal conditions. During salinity stress, RPL7 was the most stable gene according to overall ranking and the best combination of reference genes was RPL7 and RNAPol II. In contrast, GAPDH was the least stable gene which was not suitable as reference genes. The study showed that different algorithms might generate inconsistent results. Therefore, the combination of several reference genes should be selected to accurately calibrate system errors. The present study was the first to select reference genes of L. maculatus by qRT-PCR and provides a useful basis for selecting the appropriate reference gene in L. maculatus. The present study also has important implications for gene expression and functional genomics research in this species or other teleost species.

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