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

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.

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

PeerJ ◽

10.7717/peerj.5631 ◽

2018 ◽

Vol 6 ◽

pp. e5631 ◽

Cited By ~ 14

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

10.21203/rs.2.21923/v1 ◽

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.

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Evaluation of suitable reference genes in Brassica juncea and its wild relative Camelina sativa for qRT-PCR analysis under various stress conditions

PLoS ONE ◽

10.1371/journal.pone.0222530 ◽

2019 ◽

Vol 14 (9) ◽

pp. e0222530 ◽

Cited By ~ 2

Author(s):

Shikha Dixit ◽

Vinod Kumar Jangid ◽

Anita Grover

Keyword(s):

Brassica Juncea ◽

Reference Genes ◽

Camelina Sativa ◽

Stress Conditions ◽

Pcr Analysis ◽

Wild Relative ◽

Qrt Pcr ◽

Suitable Reference

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Selection of suitable reference genes for qRT-PCR analysis of Begonia semperflorens under stress conditions

Molecular Biology Reports ◽

10.1007/s11033-019-05038-5 ◽

2019 ◽

Vol 46 (6) ◽

pp. 6027-6037 ◽

Cited By ~ 1

Author(s):

Yanman Li ◽

Ying Qu ◽

Yang Wang ◽

Xue Bai ◽

Geng Tian ◽

...

Keyword(s):

Reference Genes ◽

Stress Conditions ◽

Pcr Analysis ◽

Qrt Pcr ◽

Suitable Reference ◽

Selection Of ◽

Begonia Semperflorens

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

International Journal of Molecular Sciences ◽

10.3390/ijms20010034 ◽

2018 ◽

Vol 20 (1) ◽

pp. 34 ◽

Cited By ~ 3

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.

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Selection and Stability Validation of Reference Gene Candidates for Transcriptional Analysis in Rousettus Aegyptiacus

10.21203/rs.3.rs-604945/v1 ◽

2021 ◽

Author(s):

Virginia Friedrichs ◽

Anne Balkema-Buschmann ◽

Anca Dorhoi ◽

Gang Pei

Keyword(s):

Body Temperature ◽

Reference Gene ◽

Reference Genes ◽

Core Body Temperature ◽

Transcriptional Analysis ◽

Suitable Reference Gene ◽

Type I ◽

Expression Stability ◽

Qrt Pcr ◽

Suitable Reference

Abstract Bats are the only mammals capable of powered flight and their body temperature can reach up to 42°C during flight. Additionally, bats display robust type I IFN interferon (IFN-I) responses and some species constitutively express IFN-α. Reference genes with stable expression under temperature oscillations and IFN-I release are therefore critical for normalization of quantitative reverse-transcription polymerase chain reaction (qRT-PCR) data in bats. The expression stability of reference genes in Rousettus aegyptiacus remains elusive, although this species is frequently used in the infection research. We selected ACTB, EEF1A1, GAPDH and PGK1 as candidate reference genes and evaluated their expression stability in various tissues and cells from this model bat species upon IFN-I treatment at 37°C and 40°C by qRT-PCR. We employed two statistical algorithms, BestKeeper and NormFinder, and found that EEF1A1 exhibited the highest stability under all tested conditions. ACTB and GAPDH displayed unstable expression at 40°C and upon IFN-I treatment, respectively. By normalizing to EEF1A1, we uncovered that GAPDH expression was significantly induced by IFN‑I in R. aegyptiacus. Our study identifies EEF1A1 as the most suitable reference gene for qRT-PCR studies and unveils the induction of GAPDH expression by IFN-I in R. aegyptiacus. These findings are pertinent to other bat species and even bear relevance for non-volant mammals that show physiological fluctuations of core body temperature.

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Assessment of suitable reference genes for qRT-PCR analysis in Adelphocoris suturalis

Journal of Integrative Agriculture ◽

10.1016/s2095-3119(18)61926-4 ◽

2018 ◽

Vol 17 (12) ◽

pp. 2745-2757 ◽

Cited By ~ 3

Author(s):

Jing LUO ◽

Chao MA ◽

Zhe LI ◽

Bang-qin ZHU ◽

Jiang ZHANG ◽

...

Keyword(s):

Reference Genes ◽

Pcr Analysis ◽

Qrt Pcr ◽

Suitable Reference

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Selection of Reference Genes for qRT-PCR Analysis in Lentinula edodes after Hot-Air Drying

Molecules ◽

10.3390/molecules24010136 ◽

2018 ◽

Vol 24 (1) ◽

pp. 136 ◽

Cited By ~ 3

Author(s):

Shuangshuang Gao ◽

Gangzheng Wang ◽

Zhicheng Huang ◽

Xiaoyu Lei ◽

Yinbing Bian ◽

...

Keyword(s):

Fruiting Body ◽

Reference Genes ◽

Lentinula Edodes ◽

Sulfur Compounds ◽

Pcr Analysis ◽

Expression Stability ◽

Hot Air Drying ◽

Air Drying ◽

Qrt Pcr ◽

Hot Air

Volatile sulfur compounds gradually develop in Lentinula edodes after hot-air drying, and many genes are involved in the generation of these sulfur compounds. The expression stability of reference genes may vary in a particular experimental treatment when analyzing their expressions by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, the expression profile of 17 candidate genes was assessed in L. edodes under treatment at 50 °C for 0, 1, 2, and 3 h, and the expression stability of each reference gene was analyzed by three statistical algorithms, including geNorm, NormFinder, and BestKeeper. Results indicated that the two optimal reference genes for mycelium and fruiting body were CAC and DAHP as well as CAC and NUP, respectively. Additionally, CAC and DAHP were found to be the two most stable reference genes across the mycelium and fruiting body set. Our results will provide a genetic foundation for further research on the metabolism genes of sulfur compounds in L. edodes.

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