scholarly journals Determination of the reference genes for qRT-PCR normalization and expression levels of MAT genes under various conditions in Ulocladium

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10379
Author(s):  
Li-Guo Ma ◽  
Yun Geng
Keyword(s):  
Environmental Factors ◽  
Internal Control ◽  
Dna Sequences ◽  
Reference Genes ◽  
Culture Media ◽  
Control Gene ◽  
Internal Control Gene ◽  
Type Locus ◽  
Expression Levels ◽  
Qrt Pcr

The genus Ulocladium is thought to be strictly asexual. One of the possible reasons for the lack of sexuality in Ulocladium species is the absence of the stimulus of environmental factors. Sexual reproduction in ascomycetes is controlled by a specific region in the genome referred to as mating-type locus (MAT) that consists of two dissimilar DNA sequences in the mating partners, termed MAT1-1 and MAT1-2 idiomorphs. To identify the response of MAT loci to environmental conditions, the mRNA transcription level of MAT1-1-1 and MAT1-2-1 genes was tested using qRT-PCR under different temperatures (−20 °C, −10 °C, 0 °C, 10 °C, 20 °C, 30 °C and 40 °C), culture medias (CM, OA, HAY, PCA, PDA and V8), photoperiods (24 h light, 24 h dark, 12 h light/12 h dark, 10 h light/14 h dark and 8 h light/16 h dark), and CO2 concentrations (0.03%, 0.5%, 1%, 5%, 10%, 15% and 20%). For obtaining reliable results from qRT-PCR, the most stable internal control gene and optimal number of reference genes for normalization were determined under different treatments. The results showed that there is no universal internal control gene that is expressed at a constant level under different experimental treatments. In comparison to various incubation conditions, the relative expression levels of both MAT genes were significantly increased when fungal mycelia were grown on HAY culture media at 0–10 °C with a light/dark cycle, indicating that temperature, culture media, and light might be the key environmental factors for regulating the sexuality in Ulocladium. Moreover, MAT1-1-1 and MAT1-2-1 genes showed similar expression patterns under different treatments, suggesting that the two MAT genes might play an equally important role in the sexual evolutionary process.

scholarly journals β-glucuronidase use as a single internal control gene may confound analysis in FMR1 mRNA toxicity studies

PLoS ONE ◽  
2018 ◽  
Vol 13 (2) ◽  
pp. e0192151 ◽  
Author(s):  
Claudine M. Kraan ◽  
Kim M. Cornish ◽  
Quang M. Bui ◽  
Xin Li ◽  
Howard R. Slater ◽  
...  
Keyword(s):  
Internal Control ◽  
Control Gene ◽  
Internal Control Gene ◽  
Toxicity Studies ◽  
Fmr1 Mrna

scholarly journals Custom selected reference genes outperform pre-defined reference genes in transcriptomic analysis

2019 ◽  
Author(s):  
Karen Cristine Gonçalves Dos Santos ◽  
Isabel Desgagné-Penix ◽  
Hugo Germain
Keyword(s):  
Internal Control ◽  
Reference Genes ◽  
Target Genes ◽  
Differential Expression Analysis ◽  
Effector Proteins ◽  
Sequencing Data ◽  
Library Size ◽  
Expression Levels ◽  
Expression Arrays ◽  
Internal Control Genes

Abstract Background: RNA sequencing allows the measuring of gene expression at a resolution unmet by expression arrays or RT-qPCR. It is however necessary to normalize sequencing data by library size, transcript size and composition, among other factors, before comparing expression levels. The use of internal control genes or spike-ins is advocated in the literature for scaling read counts, but the methods for choosing reference genes are mostly targeted at RT-qPCR studies and require a set of pre-selected candidate controls or pre-selected target genes. Results: Here, we report an R-based script to select internal control genes based solely on read counts and gene sizes. This novel method first normalizes the read counts to Transcripts per Million (TPM) and then excludes weakly expressed genes using the DAFT script to calculate the cut-off. It then selects as references the genes with lowest TPM covariance. We used this method to pick custom reference genes for the differential expression analysis of three transcriptome sets from transgenic Arabidopsis plants expressing heterologous fungal effector proteins tagged with GFP (using GFP alone as the control). The custom reference genes showed lower covariance and fold change as well as a broader range of expression levels than commonly used reference genes. When analyzed with NormFinder, both typical and custom reference genes were considered suitable internal controls, but the custom selected genes were more stable. geNorm produced a similar result in which most custom selected genes ranked higher (i.e. were more stable) than commonly used reference genes. Conclusions: The proposed method is innovative, rapid and simple. Since it does not depend on genome annotation, it can be used with any organism, and does not require pre-selected reference candidates or target genes that are not always available.

scholarly journals Selection of Reference Genes for qRT-PCR in Bletilla striata under Heat Stress

2021 ◽  
Vol 25 (03) ◽  
pp. 547-554
Author(s):  
Fang Liang
Keyword(s):  
Internal Control ◽  
Reference Genes ◽  
Elongation Factor ◽  
Pcr Analysis ◽  
Bletilla Striata ◽  
Traditional Chinese Herbal Medicine ◽  
Qrt Pcr ◽  
Wide Range ◽  
Root Tuber ◽  
Different Tissues

Bletilla striata (Thunb.) Reihb.f., a traditional Chinese herbal medicine, has attracted increasing attention because of its wide range of applicability to the medical field and chemical industry. B. striata has been identified to be particularly sensitive to high temperatures. Thus, the study of temperature stress on gene transcription is of interest in the field. Use of reliable reference genes is essential for qRT-PCR analysis of genes. However, little information regarding suitable reference genes for the genus Bletilla has been published. In this study, the sequences of seven potential reference genes in B. striata were obtained via a homology cloning strategy. These genes were as follows: glyceraldehydes-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), α-tubulin (TUA), β-tubulin (TUB), ubiquitin (UBI), and NAC domain protein (NAC). We then evaluated the stability levels of these transcripts in different tissues (root, tuber, and leaf) exposed to high temperature using three conventional software and comprehensive RefFinder algorithms. The results indicated that 18S and TUB were the best internal control genes among different periods of heat treatment and that a combination of 18S and UBI was the best in different tissues. Altogether, 18S and UBI were identified to be the best reference genes for all the samples, while NAC and TUA were the least stable reference genes. The results will be useful for studies on target gene expression in plants of the Bletilla genus. © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers © 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers© 2021 Friends Science Publishers©

scholarly journals Custom selected reference genes outperform pre-defined reference genes in transcriptomic analysis

2019 ◽  
Author(s):  
Karen Cristine Gonçalves Dos Santos ◽  
Isabel Desgagné-Penix ◽  
Hugo Germain
Keyword(s):  
Internal Control ◽  
Reference Genes ◽  
Target Genes ◽  
Differential Expression Analysis ◽  
Effector Proteins ◽  
Sequencing Data ◽  
Library Size ◽  
Expression Levels ◽  
Expression Arrays ◽  
Internal Control Genes

Abstract Background: RNA sequencing allows the measuring of gene expression at a resolution unmet by expression arrays or RT-qPCR. It is however necessary to normalize sequencing data by library size, transcript size and composition, among other factors, before comparing expression levels. The use of internal control genes or spike-ins is advocated in the literature for scaling read counts, but the methods for choosing reference genes are mostly targeted at RT-qPCR studies and require a set of pre-selected candidate controls or pre-selected target genes. Results: Here, we report an R-based script to select internal control genes based solely on read counts and gene sizes. This novel method first normalizes the read counts to Transcripts per Million (TPM) and then excludes weakly expressed genes using the DAFS script to calculate the cut-off. It then selects as references the genes with lowest TPM covariance. We used this method to pick custom reference genes for the differential expression analysis of three transcriptome sets from transgenic Arabidopsis plants expressing heterologous fungal effector proteins tagged with GFP (using GFP alone as the control). The custom reference genes showed lower covariance and fold change as well as a broader range of expression levels than commonly used reference genes. When analyzed with NormFinder, both typical and custom reference genes were considered suitable internal controls, but the expression of custom selected genes was more stable. geNorm produced a similar result in which most custom selected genes ranked higher (i.e. expression more stable) than commonly used reference genes. Conclusions: The proposed method is innovative, rapid and simple. Since it does not depend on genome annotation, it can be used with any organism, and does not require pre-selected reference candidates or target genes that are not always available.

Identification of suitable reference genes of Scylla paramamosain for gene expression profiling in various tissues and under vibrio challenge

Crustaceana ◽  
2018 ◽  
Vol 91 (10) ◽  
pp. 1195-1210 ◽  
Author(s):  
Yabo Fang ◽  
Le Diao ◽  
Fengying Zhang ◽  
Lingbo Ma ◽  
Mengdi Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
18S Rrna ◽  
Elongation Factor ◽  
Scylla Paramamosain ◽  
Mud Crab ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Elongation Factor 1

Abstract The quantitative real-time transcription-polymerase chain reaction (qRT-PCR) is now used widely in studies about mRNA expression levels. The selection of one or more stable reference gene(s) used for data normalization is substantial. In this study, expression levels of eleven candidate reference genes (β-actin, 16S rRNA, 18S rRNA, 28S rRNA, α-I tubulin, GAPDH, ribosomal protein L13, elongation factor 1 α, elongation factor 2, arginine kinase and ubiquitin) were examined using the GenomeLab GeXP analysis system (Beckman Coulter). Gene expression data were analysed using two different statistical models: geNorm and NormFinder. (1) In six different tissues (hepatopancreas, haemocytes, heart, gill, muscle, and testis) from the mud crab, Scylla paramamosain, 18S rRNA and elongation factor 1 α were identified as the two best reference genes. (2) In the haemocytes after being challenged by Vibro parahaemolyticus, the result suggested that ubiquitin was the most stable gene after the treatment. 18S rRNA, elongation factor 1 α and ubiquitin are herein recommended as the best combination. These results provide useful options for reference gene selection under different experimental conditions in qRT-PCR studies in the mud crab.

Comparison of ABL1 and Gusb Reference Genes in the qRT-PCR Analysis of Halving Time and Early Molecular Response to TKI Therapy in Patients with Chronic Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5424-5424 ◽  
Author(s):  
Joanna Wącław ◽  
Magdalena Zawada ◽  
Sylwia Czekalska ◽  
Bogdan Ochrem ◽  
Tomasz Sacha
Keyword(s):  
Predictive Factor ◽  
Reference Genes ◽  
Chronic Phase ◽  
Imatinib Therapy ◽  
Pcr Analysis ◽  
Molecular Response ◽  
Control Gene ◽  
Blood Samples ◽  
Transcript Levels ◽  
Qrt Pcr

Abstract Objectives and background: Early molecular response (EMR) to tyrosine kinase inhibitors (TKI) therapy defined as BCR-ABL1 transcript level ≤ 10% at 3 months is a well-established predictive factor in CML patients. Recently, several study groups presented the concept of BCR-ABL1 decline velocity or "halving time" as a new predictive factor for optimal TKI treatment response. Different reference genes were used in the quantitative RT-PCR (qRT-PCR) analysis to calculate halving time and EMR rate, potentially influencing results of these analyses. The aim of the study was to compare ABL1 and beta-glucuronidase (GUSB) as reference genes used for calculation of halving time and EMR rate in chronic phase CML patients (pts) treated with imatinib 400 mg/d in the clinical settings. Methods: A total of 52 chronic phase CML pts with blood samples collected at baseline and at 3 months of imatinib therapy were investigated. Transcript levels of BCR-ABL1 were determined by qRT-PCR with the use of ABL1 and GUSB as control genes for each patient. Halving time for BCR-ABL1 transcripts was calculated using baseline transcript levels, transcript levels at 3 months of treatment, and number of days between these two points according to methodology reported elswhere1. Using receiver operating characteristic (ROC) analysis, the optimal halving time thresholds for achievement of EMR for each control gene were determined. Optimal thresholds along the ROC curves were calculated using the Youden index. Results: 40 of 52 (76.9%) and 44 of 52 (84.6%) pts achieved EMR with BCR-ABL1 transcripts levels calculated with GUSB and ABL1 as control genes, respectively (p=0.55). In 4 pts there was no BCR-ABL1 transcript reduction from baseline at 3 month using GUSB as a control gene resulting with negative halving times. The GUSB based halving times of these 4 patients were imputed to the longest positive halving time (8530 days) calculated for the patient with the smallest BCR-ABL1 decline. Transcript reduction occurred in all pts when ABL1 was used as a control gene. ROC curve analysis resulted in the optimal halving time thresholds for achievement of EMR of 39 days (area under the curve [AUC ] 0.96, ;95% CI 0.9-1.0, ) and 31 days (AUC 0.95; 95% CI 0.87-1.0, ) for GUSB and ABL1 control gene, respectively. Discussion and Conclusions: Lack of linearity in qRT-PCR analysis observed in blood samples collected in CML patients at diagnosis, with high amount of BCR-ABL1 and wild type ABL1 transcripts could affect the accuracy of early kinetics decline of BCR-ABL1 transcript calculations when ABL1 is used as a reference gene. The analysis with the use of GUSB as a control gene is not biased by this phenomenon. In this study, a non - statistically significant differences in the rate of EMR achieved at 3 months of imatinib therapy and halving time were observed. Analyses performed in our study with ABL1 and GUSB as reference genes suggest that both GUSB and ABL1 are able to identify relevant cut-offs for EMR achievement and could be used for halving time calculation. Reference:Branford S, Yeung DT, Parker WT, et al. Prognosis for patients with CML and >10% BCR-ABL1 after 3 months of imatinib depends on the rate of BCR-ABL1 decline. Blood 2014 Jul 24;124(4):511-8 Disclosures Zawada: Novartis: Speakers Bureau. Czekalska:Novartis: Speakers Bureau. Sacha:Pfizer: Consultancy, Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Adamed: Consultancy, Honoraria.

β-Glucuronidase is a suitable internal control gene for mRNA quantitation in pathophysiological and non-pathological livers

2013 ◽  
Vol 95 (2) ◽  
pp. 131-135 ◽  
Author(s):  
Hiromi Yamaguchi ◽  
Sawako Matsumoto ◽  
Mariko Ishibashi ◽  
Kiyoshi Hasegawa ◽  
Masahiko Sugitani ◽  
...  
Keyword(s):  
Internal Control ◽  
Control Gene ◽  
Internal Control Gene ◽  
Mrna Quantitation

Selection of the Internal Control Gene for Real-Time Quantitative RT-PCR Assays in Temperature Treated Leptospira

2008 ◽  
Vol 56 (6) ◽  
pp. 539-546 ◽  
Author(s):  
Erika Margarita Carrillo-Casas ◽  
Rigoberto Hernández-Castro ◽  
Francisco Suárez-Güemes ◽  
Alejandro de la Peña-Moctezuma
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Control Gene ◽  
Internal Control Gene ◽  
Rt Pcr ◽  
Pcr Assays ◽  
Selection Of

scholarly journals Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Min-dong Chen ◽  
Bin Wang ◽  
Yong-ping Li ◽  
Mei-juan Zeng ◽  
Jian-ting Liu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Expression Patterns ◽  
Suitable Reference Gene ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Suitable Reference ◽  
Gene Expression Levels

AbstractSelecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

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