scholarly journals Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization inIris. lacteavar.chinensisRoots under Cadmium, Lead, and Salt Stress Conditions

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Chun-Sun Gu ◽  
Liang-qin Liu ◽  
Chen Xu ◽  
Yan-hai Zhao ◽  
Xu-dong Zhu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Salt Stress ◽  
Real Time ◽  
Reference Genes ◽  
Elongation Factor ◽  
Stress Conditions ◽  
Translation Initiation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Wide Range

Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1α), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), inIris. lacteavar.chinensis(I. lacteavar.chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (Ct) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed thatEIF-5AandUBCwere the most stable reference genes across all of the tested samples, whileTUBLINwas unsuitable as internal controls.I. lacteavar.chinensisis tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses.

scholarly journals Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail, Lymnaea stagnalis

2019 ◽  
Author(s):  
Alexander P Young ◽  
Carmen F Landry ◽  
Daniel J Jackson ◽  
Russell C Wyeth
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Lymnaea Stagnalis ◽  
Elongation Factor ◽  
Pond Snail ◽  
Tissue Samples ◽  
Systematic Assessment ◽  
Wide Range ◽  
The Stability ◽  
Suitable Reference

Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression across multiple tissues. To obtain reliable results, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail (Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging target for qPCR experimentation. However, no systematic assessment of reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from a variety of tissues in L. stagnalis. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin (ACTB), beta-tubulin (TUBB), ubiquitin (UBI), prenylated rab acceptor protein 1 (Rapac1), and a voltage gated potassium channel (VGKC). These genes exhibited a wide range of expression levels among tissues. The stability of each of the genes was consistent when measured by any of the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper and RefFinder. Our data indicate that GAPDH and EF1α are highly stable in the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle) as well as in pooled analyses. We do not recommend VGKC for use in RT-qPCR experiments due to its relatively low expression stability. Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we suggest GAPDH and EF1α are a strong option for multi-tissue analyses of RT-qPCR data in Lymnaea stagnalis.

scholarly journals Validation of Real-time PCR Reference Genes of Muscle Metabolism in Harvested Spiny-Cheek Crayfish (Faxonius limosus) Exposed to Seasonal Variation

Animals ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1140
Author(s):  
Natalia Śmietana ◽  
Remigiusz Panicz ◽  
Małgorzata Sobczak ◽  
Piotr Eljasik ◽  
Przemysław Śmietana
Keyword(s):  
Real Time ◽  
Calcium Binding ◽  
Reference Genes ◽  
Target Genes ◽  
Arginine Kinase ◽  
Elongation Factor ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Systematic Analysis ◽  
Key Species

Real-time quantitative reverse transcription PCR (RT-qPCR) is a sensitive and broadly used technique of assessing gene activity. To obtain a reliable result, stably expressed reference genes are essential for normalization of transcripts in various samples. To our knowledge, this is the first systematic analysis of reference genes for normalization of RT-qPCR data in spiny-cheek crayfish (Faxonius limosus). In this study, expression of five candidate reference genes (actb, β-actin; gapdh, glyceraldehyde-3-phosphate dehydrogenase; eif, eukaryotic translation initiation factor 5a; ef-1α, elongation factor-1α; and tub, α-tubulin) in muscle samples from male and female F. limosus in spring and autumn was analyzed. Additionally, the most stable reference genes were used for accurate normalization of five target genes, i.e., tnnc, troponin c; ak, arginine kinase; fr, ferritin; ccbp-23, crustacean calcium-binding protein 23; and actinsk8, skeletal muscle actin 8. Results obtained using the geNorm and NormFinder algorithms showed high consistency, and differences in the activity of the selected actb with eif genes were successfully identified. The spring and autumn activities of the target genes (except ak) in the muscle tissue of males and females differed significantly, showing that both sexes are immensely involved in an array of breeding behaviors in spring, and females intensively recover in the autumn season. Characterization of first reference genes in spiny-cheek crayfish will facilitate more accurate and reliable expression studies in this key species.

scholarly journals Transcriptome-based identification and validation of optimal reference genes for quantitative real-time PCR normalization in Psathyrostachys huashanica

2020 ◽  
Author(s):  
Chuan Shen ◽  
Jingyuan Li ◽  
Caiyan Wei ◽  
Xudong Zhang ◽  
Yunfeng Wu
Keyword(s):  
Gene Expression ◽  
Abiotic Stress ◽  
Real Time ◽  
Temperature Stress ◽  
Reference Genes ◽  
18S Rrna ◽  
Stress Conditions ◽  
Psathyrostachys Huashanica ◽  
Biotic And Abiotic Stress ◽  
Ubiquitin Conjugating Enzyme

Abstract Background: P. huashanica ( Psathyrostachys huashanica ), known as an important resistance resource reservoir, is a rare and endangered plant growing suitably in Huashan mount region and would be urgently exploited in wheat genetic improvements sooner. During the utilization process, different IRGs (internal reference genes) need to be appropriately selected as standards based on biotic and abiotic stress conditions. It is crucial that Real-time RT-qPCR with combination of bioinformatics were adopted to explore the reliable IRGs from transcriptome of P . huashanica.Results: The present work reported new 3 species of IRGs, UBC2 , UBC17, 18S rRNA , which were screened from transcriptome of P. huashanica under biotic and abiotic stress conditions, using RT-qPCR and four algorithms, including geNorm, NormFinder, BestKeeper, and RefFinder, to analyse expression of sixteen candidate reference genes. These genes appear as following 18S rRNA (18S ribosomal RNA), EF1-α (eukaryotic elongation factor 1 alpha), UBC2 (ubiquitin-conjugating enzyme E2-2), UBC17 (ubiquitin-conjugating enzyme E2-17), α-TUB2A (alpha tubulin-2A), β-TUB3 (beta tubulin 3), ADF4 (Actin-depolymerising factor 4), ACTIN (actin), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), 60SARP (60S acidic ribosomal protein), UBQ (polyubiquitin), SamDC (S-Adenosylmethionine decarboxylase), EIF4A (eukaryotic initiation factor 4A), ARF (ADP-ribosylation factor), HIS1 (histone H1), and HIS2B (histone H2B). Analysis of gene expression demonstrated that the expression of UBC2 gene was most stable under ABA hormone stress, low temperature stress and high temperature stress, similarly, UBC17 gene under IAA hormone stress, salinity stress and drought stress, both UBC17 genes and 18S rRNA genes under abiotic and biotic stress, respectively. The most stable gene was UBC2 gene in the root, UBC17 gene in stem and leaf. In this study, α-TUB2A , UBC and ACTIN genes were verified as the suitable reference genes across all tested samples. To further validate the suitability of the selected reference genes, we evaluated the relative expression of PsaCPK3 (Calcium-dependent protein kinase) and PsaHSP70-1 (heat shock protein 70-1), which are stress-related genes that may be involved in response to adversity.Conclusions: This study has identified a set of the most stable IRGs suiting for RT-qPCR detection of a few target gene expressions from P . huashanica in different experimental conditions. In addition, this study should provide the accuracy information for gene expression analysis in P . huashanica .

Phylogeny and morphology reveal two new species of Diaporthe from Betula spp. in China

Phytotaxa ◽  
2016 ◽  
Vol 269 (2) ◽  
pp. 90 ◽  
Author(s):  
ZHUO DU ◽  
XIN-LEI FAN ◽  
KEVIN D. HYDE ◽  
QIN YANG ◽  
YING-MEI LIANG ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Sequence Data ◽  
Histone H3 ◽  
Elongation Factor ◽  
Betula Platyphylla ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Wide Range ◽  
Dieback Disease ◽  
Elongation Factor 1

Diaporthe species are common pathogens, endophytes, or saprobes on a wide range of hosts. During our investigation of forest pathogens, we made collections of Diaporthe species associated with canker and dieback disease of Betula platyphylla and B. albosinensis in Sichuan and Shaanxi provinces in China. Diaporthe betulae sp. nov. and D. betulicola sp. nov. are introduced in this paper, with illustrations, descriptions and support from analysis of ribosomal DNA internal transcribed spacer (ITS), calmodulin (CAL), histone H3 (HIS), translation elongation factor 1-α (TEF1-α) and beta-tubulin (TUB2) sequence data. Diaporthe betulae is characterized by hyaline, ellipsoidal, aseptate, biguttulate, 8.5–11 × 3–4 µm alpha conidia. Diaporthe betulicola is characterized by pycnidial stromata with a single locule with one ostiole per disc. Alpha conidia are hyaline, oblong, aseptate, lack guttules and 9.9–14.7 × 1.3–2.5 µm, and beta conidia are hyaline, spindle-shaped, curved, aseptate and 17–24 × 0.7–1.2 µm.

scholarly journals Characterization of the translation elongation factor 1-α gene in a wide range of pathogenic Aspergillus species

2017 ◽  
Vol 66 (4) ◽  
pp. 419-429 ◽  
Author(s):  
Sadegh Nouripour-Sisakht ◽  
Bahram Ahmadi ◽  
Koichi Makimura ◽  
Sybren de Hoog ◽  
Yoshiko Umeda ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Aspergillus Species ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Wide Range ◽  
Elongation Factor 1

scholarly journals Selection of reliable reference genes for quantitative RT-PCR in garlic under salt stress

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7319 ◽  
Author(s):  
Guanglong Wang ◽  
Chang Tian ◽  
Yunpeng Wang ◽  
Faxiang Wan ◽  
Laibao Hu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Salt Stress ◽  
Reference Genes ◽  
Expression Profiles ◽  
Accurate Determination ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Specific Reference ◽  
Variable Expression ◽  
Selection Of

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) has been frequently used for detecting gene expression. To obtain reliable results, selection of suitable reference genes is a fundamental and necessary step. Garlic (Allium sativum), a member from Alliaceae family, has been used both as a food flavoring and as a traditional medicine. In the present study, garlic plants were exposed to salt stress (200 mM NaCl) for 0, 1, 4 and 12 h, and garlic roots, bulbs, and leaves were harvested for subsequent analysis. The expression stability of eight candidate reference genes, eukaryotic translation initiation factor 4α (eIF-4α), actin (ACTIN), tubulin β-7 (TUB7), TAP42-interacting protein of 41 kDa (TIP41), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), SAND family protein (SAND), elongation factor 1 alpha (EF-1α), and protein phosphatase 2A (PP2A) were evaluated by geNorm, NormFinder, and BestKeeper. All genes tested displayed variable expression profiles under salt stress. In the leaf and root group, ACTIN was the best reference gene for normalizing gene expression. In garlic clove, ACTIN and SAND were the least variable, and were suitable for gene expression studies under salt stress; these two genes also performed well in all samples tested. Based on our results, we recommend that it is essential to use specific reference genes in different situations to obtain accurate results. Using a combination of multiple stable reference genes, such as ACTIN and SAND, to normalize gene expression is encouraged. The results from the study will be beneficial for accurate determination of gene expression in garlic and other plants.

scholarly journals Characterization of Diaporthe species associated with peach constriction canker, with two novel species from China

MycoKeys ◽  
2021 ◽  
Vol 80 ◽  
pp. 77-90
Author(s):  
Xianhong Wang ◽  
Yashuang Guo ◽  
Yamin Du ◽  
Ziling Yang ◽  
Xinzhong Huang ◽  
...  
Keyword(s):  
Internal Transcribed Spacer ◽  
Novel Species ◽  
Phylogenetic Analyses ◽  
Elongation Factor ◽  
Translation Elongation Factor ◽  
Translation Elongation ◽  
Wide Range ◽  
Elongation Factor 1

Species of Diaporthe infect a wide range of plants and live in vivo as endophytes, saprobes or pathogens. However, those in peach plants are poorly characterized. In this study, 52 Diaporthe strains were isolated from peach branches with buds, showing constriction canker symptoms. Phylogenetic analyses were conducted using five gene regions: internal transcribed spacer of the ribosomal DNA (ITS), translation elongation factor 1-α (TEF), ß-tubulin (TUB), histone (HIS), and calmodulin (CAL). These results coupled with morphology revealed seven species of Diaporthe, including five known species (D. caryae, D. cercidis, D. eres, D. hongkongensis, and D. unshiuensis). In addition, two novel species D. jinxiu and D. zaofenghuang are introduced. Except for the previously reported D. eres, this study represents the first characterization of Diaporthe species associated with peach constriction canker in China, and contributes useful data for practicable disease management.

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