Stable Reference Gene Selection for qRT-PCR Normalization in Strawberry (Fragaria × ananassa) Leaves under Different Stress and Light-Quality Conditions

Selecting an appropriate reference gene is of crucial importance for improving the accuracy of qRT-PCR analyses. In this study, strawberry (Fragaria ananassa) seedlings were subjected to different environmental conditions including heat, cold, drought, salt, white-light, blue-light, and red-light treatments. The expression levels of seven candidate reference genes, including Fa18S, FaGAPDH, FaPIRUV, FaDBP, FaHISTH4, FaACTIN1, and FaACTIN2, in the strawberry leaves were measured by qRT-PCR. Then, four programs (geNorm, NormFinder, BestKeeper, and RefFinder) were employed as tools to evaluate the expression stability of the candidate reference genes. The results showed that the expression stability of the reference genes varied under different conditions. For the cold stress and white-light treatments, FaACTIN2 was evaluated to be the most stable reference gene. FaGAPDH should be used as the reference gene under salt-stress condition and red-light treatment. For the data normalization under drought-stress treatment, FaDBP is the recommended reference gene with the highest expression stability. FaHISTH4 was observed to be the best reference gene for data normalization under heat stress and blue-light treatment. This work provides information on selecting reference genes for accurate gene expression analyses of target genes in strawberry leaves under various abiotic stress and light-quality conditions.

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

Genes ◽

10.3390/genes10090647 ◽

2019 ◽

Vol 10 (9) ◽

pp. 647 ◽

Cited By ~ 4

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.

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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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Selection and stability validation of reference gene candidates for transcriptional analysis in Rousettus aegyptiacus

Scientific Reports ◽

10.1038/s41598-021-01260-z ◽

2021 ◽

Vol 11 (1) ◽

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

AbstractBats 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 35 °C, 37 °C and 40 °C by qRT-PCR. We employed two statistical algorithms, BestKeeper and NormFinder, and found that EEF1A1 exhibited the highest expression stability under all tested conditions. ACTB and GAPDH displayed unstable expression upon temperature change and 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 upon temperature changes and IFN-I treatment and unveils the induction of GAPDH expression by IFN-I in R. aegyptiacus. These findings are pertinent to other bat species and may be relevant for non-volant mammals that show physiological fluctuations of core body temperature.

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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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Selection and Validation of Appropriate Reference Genes for Gene Expression Studies in Schima Superba

10.21203/rs.3.rs-145449/v1 ◽

2021 ◽

Author(s):

Zhongyi Yang ◽

Rui Zhang ◽

Zhichun Zhou

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Geometric Mean ◽

Gene Transcript ◽

Expression Stability ◽

Schima Superba ◽

Expression Studies ◽

Gene Expression Studies ◽

Different Tissues

Abstract Background Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

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Identification and expression of the trehalose-6-phosphate synthase gene family members in tomato exposed to different light spectra

Archives of Biological Sciences ◽

10.2298/abs160325082c ◽

2017 ◽

Vol 69 (1) ◽

pp. 93-101

Author(s):

Zexiong Chen ◽

Juan Lou

Keyword(s):

Plant Growth ◽

Blue Light ◽

White Light ◽

Growth And Development ◽

Light Quality ◽

Red Light ◽

Floral Transition ◽

Stem Length ◽

Pcr Analysis ◽

Phosphate Synthase

Light is the source of energy for plants. Light wavelengths, densities and irradiation periods act as signals directing morphological and physiological characteristics during plant growth and development. To evaluate the effects of light wavelengths on tomato growth and development, Solanum lycopersicum (cv. micro-Tom) seedlings were exposed to different light-quality environments, including white light and red light supplemented with blue light (at ratios of 3:1 and 8;1, respectively). Tomatoes grown under red light supplemented with blue light displayed significantly shorter stem length, a higher number of flower buds and rate of fruit set, but an extremely late flowering compared to white-light-grown plants. To illustrate the mechanism underlying the inhibition of stem growth and floral transition mediated by red/blue light, 10 trehalose-6-phosphate synthase (TPS) genes were identified in tomato, and bioinformatics analysis was performed. qRT-PCR analysis showed that SlTPSs were expressed widely throughout plant development and SlTPS1 was expressed at extremely high levels in stems and buds. Further analysis of several flowering-associated genes and microRNAs showed that the expressions of SlTPS1, SlFT and miR172 were significantly downregulated in tomato grown under red and blue light compared with those grown under white light, whereas miR156 transcript levels were increased. A regulatory model underlying vegetative growth and floral transition regulated by light qualities is presented. Our data provide evidence that light quality strongly affects plant growth and phase transition, most likely via the TPS1-T6P signaling pathway.

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RT-qPCR analyses on the osteogenic differentiation from human iPS cells: An investigation of reference genes

10.1101/2020.05.27.118000 ◽

2020 ◽

Author(s):

Kensuke Okamura ◽

Yusuke Inagaki ◽

Takeshi K. Matsui ◽

Masaya Matsubayashi ◽

Tomoya Komeda ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Reference Gene ◽

Reference Genes ◽

Ips Cells ◽

Expression Stability ◽

Differentiation Markers ◽

Osteogenic Differentiation Medium ◽

Need To Evaluate ◽

Induced Pluripotent ◽

Beta 2 Microglobulin

AbstractReverse transcription quantitative PCR (RT-qPCR) is used to quantify gene expression and require standardization with reference genes. We sought to identify the reference genes best suited for experiments that induce osteogenic differentiation from human induced pluripotent stem (iPS) cells. They were cultured in an undifferentiated maintenance medium and after confluence, further cultured in an osteogenic differentiation medium for 28 days. RT-qPCR was performed on undifferentiation markers, osteoblast and osteocyte differentiation markers, and reference gene candidates. The expression stability of each reference gene candidate was ranked using four algorithms. General rankings identified TATA box binding protein (TBP) in the first place, followed by transferrin receptor (TFRC), ribosomal protein large P0 (RPLP0), and finally, beta-2-microglobulin (B2M), which was revealed as the least stable. Interestingly, universally used GAPDH and ACTB were found to be unsuitable. Our findings strongly suggest a need to evaluate the expression stability of reference gene candidates for each experiment.

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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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Arachidonic acid in the lipids of marine algae maintained under blue, white and red light

Zeitschrift für Naturforschung C ◽

10.1515/znc-1988-1-205 ◽

1988 ◽

Vol 43 (1-2) ◽

pp. 15-18 ◽

Cited By ~ 4

Keyword(s):

Fatty Acids ◽

Arachidonic Acid ◽

White Light ◽

Marine Algae ◽

Light Quality ◽

Red Light ◽

Major Constituent ◽

Enteromorpha Intestinalis ◽

Definite Effect

Marine algae, maintained for one month under blue, white and red light were rather rich in lipids but obviously poor in fat (triacylglycerols). These lipids consisted predominantly of glycolipids and phospholipids. Irrespective of the light quality, the major constituent fatty acids in lipids of these algae were, in most cases, those with 20 carbon atoms. The light quality had a definite effect on the proportion of arachidonic acid in the lipids of certain algae. Thus, the proportion of arachidonic acid in Enteromorpha intestinalis maintained under white light was 45% and in Sargassum salicifolium kept under red light 25% of the total constituent fatty acids in the total algal lipids

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Die Wirkung von Phytochrom auf die Bildung von Einzelcarotinoiden in etiolierten Horde um -Keimlingen / The Influence of Phytochrome on the Formation of Individual Carotenoids in Etiolated Hordeum Seedlings

Zeitschrift für Naturforschung C ◽

10.1515/znc-1975-1-214 ◽

1975 ◽

Vol 30 (1-2) ◽

pp. 67-68 ◽

Cited By ~ 10

Author(s):

Hans K. Kleudgen ◽

Hartmut K. Lichtenthaler

Keyword(s):

White Light ◽

Accumulation Rate ◽

Red Light ◽

Light Treatment ◽

Light Effects ◽

Short Time ◽

Β Carotene

Abstract Short time red pulses, given 6 times for 5 min within 36 h, induce in etiolated barley seedlings an enhanced synthesis of the main chloroplast carotenoids β-carotene, violaxanthine, lutein and neoxanthine. The level of antheraxanthine and zeaxanthine decreases by red light treatment. These red light effects are reverted by subsequent short time far-red pulses. The results show that the white light induced change in the accumulation rate of individual carotenoids is initiated and regulated by active phytochrome Pfr . In the case of neoxanthin and zeaxanthin the red light effects cannot be fully reverted by far-red; this points to very fast phytochrome reaction.

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