scholarly journals Reference gene screening of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans for quantitative real-time PCR studies

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Elin Verbrugghe ◽  
Frank Pasmans ◽  
An Martel
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Batrachochytrium Dendrobatidis ◽  
Expression Patterns ◽  
Growth Stages ◽  
Batrachochytrium Salamandrivorans ◽  
The Stability ◽  
Different Growth Stages

AbstractReal-time quantitative PCR studies largely depend on reference genes for the normalization of gene expression. Stable reference genes should be accurately selected in order to obtain reliable results. We here present a study screening commonly used reference genes (TEF1F, α-centractin, Ctsyn1, GAPDH, R6046, APRT and TUB) in the chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), which cause the lethal amphibian skin disease chytridiomycosis. We evaluated the stability of the reference gene candidates during different growth stages of the fungi, using different statistical software packages: ΔCT, BestKeeper, GeNorm, NormFinder and RefFinder. In order to reflect the in vivo situation, the stability of the candidates was assessed when taking all growth stages into account. Using an ex-vivo approach, we tested whether the expression of GAPDH, TUB, R6046 and APRT (Bd) and GAPDH, TUB, R6046 and α-centractin (Bsal) remained stable when these fungi came in contact with host tissue. Finally, their role as in vivo reference genes was examined in skin tissue of experimentally infected midwife toads (Alytes obstetricans) (Bd) and fire salamanders (Salamandra salamandra) (Bsal). Summarized, the present study provides guidance for selecting appropriate reference genes when analyzing expression patterns of these fungal organisms during different growth stages and in Bd- or Bsal-infected tissues.

scholarly journals Optimizations for identifying reference genes in bone and cartilage bioengineering

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fei Xiong ◽  
Xiangyun Cheng ◽  
Chao Zhang ◽  
Roland Manfred Klar ◽  
Tao He
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Adipose Tissue ◽  
Real Time ◽  
Reference Gene ◽  
Muscle Tissue ◽  
Reference Genes ◽  
Target Genes ◽  
The Stability ◽  
And Cartilage

Abstract Background Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering. Results The stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes. Conclusions Based on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.

scholarly journals Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR in Macrobrachium Nipponense

2018 ◽  
Vol 19 (8) ◽  
pp. 2258 ◽  
Author(s):  
Yuning Hu ◽  
Hongtuo Fu ◽  
Hui Qiao ◽  
Shengming Sun ◽  
Wenyi Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Translation Initiation Factor ◽  
Suitable Reference Gene ◽  
Quantitative Real Time Pcr ◽  
Macrobrachium Nipponense ◽  
The Stability ◽  
Suitable Reference

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

scholarly journals IDENTIFICATION AND VALIDATION OF REFERENCE GENES FOR THE NORMALIZATION IN REAL-TIME RT-QPCR ON RICE AND RED RICE IN COMPETITION, UNDER DIFFERENT NITROGEN DOSES

2017 ◽  
Vol 35 (0) ◽  
Author(s):  
D.P. BENEMANN ◽  
A.M. NOHATO ◽  
L. VARGAS ◽  
L.A AVILA ◽  
D. AGOSTINETTO
Keyword(s):  
Real Time ◽  
Reference Gene ◽  
Analysis Of Variance ◽  
Reference Genes ◽  
Target Gene ◽  
Red Rice ◽  
Nitrogen Levels ◽  
Cytosolic Ascorbate Peroxidase ◽  
Polymerase Chain ◽  
The Stability

ABSTRACT Real time reverse transcription polymerase chain reaction (RT-qPCR) is an important technique to analyze differences in gene expression due to its sensitivity, accuracy, and specificity. However, before analyzing the expression of the target gene, it is necessary to identify and evaluate the stability of candidate reference genes for the proper normalization. This study aimed at evaluating the stability of candidate reference genes in order to identify the most appropriate genes for the normalization of the transcription in rice and red rice in competition under different nitrogen levels, as well as to demonstrate the effectiveness of the reference gene selected for the expression of the cytosolic ascorbate peroxidase (OsAPX2). Eleven candidate reference genes were assessed using the RefFinder which integrates the four leading software: geNorm, NormFinder, Bestkeeper, and the comparative delta-Ct method in addition to the analysis of variance to identify genes with lower standard deviation and coefficient of variation values. Eight out of the eleven genes have shown the desired effectiveness and, among them, the gene UBC-E2 has the highest stability according to RefFinder and the analysis of variance. The expression of the gene OsAPX2 has proven to be effective in validating the candidate reference gene. This study is the first survey on the stability of candidate reference genes in rice and red rice in competition, providing information to obtain more accurate results in RT-qPCR.

scholarly journals Selection of reference genes for quantitative real-time polymerase chain reaction in porcine embryos

2017 ◽  
Vol 29 (2) ◽  
pp. 357 ◽  
Author(s):  
Won-Jae Lee ◽  
Si-Jung Jang ◽  
Seung-Chan Lee ◽  
Ji-Sung Park ◽  
Ryoung-Hoon Jeon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
In Vitro Fertilisation ◽  
Accurate Normalisation ◽  
Study Gene Expression ◽  
Succinate Dehydrogenase Complex ◽  
The Stability

To study gene expression and to determine distinctive characteristics of embryos produced by different methods, normalisation of the gene(s) of interest against reference gene(s) has commonly been employed. Therefore, the present study aimed to assess which reference genes tend to express more stably in single porcine blastocysts produced in vivo (IVO) or by parthenogenetic activation (PA), in vitro fertilisation (IVF) and somatic cell nuclear transfer (SCNT) using different analysis programs, namely geNorm, Normfinder and Bestkeeper. Commonly used reference genes including 18S rRNA (18S), H2A histone family, member Z (H2A), hypoxanthine phosphoribosyltransferase1 (HPRT1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein 4 (RPL4), peptidylprolyl isomerase A (PPIA), beta actin (ACTB), succinate dehydrogenase complex, subunit A (SDHA) and hydroxymethylbilane synthase (HMBS2) were analysed; most of them resulted in significantly (P < 0.05) different cycle threshold (CT) values in porcine embryos except for SDHA and H2A. In evaluation of stable reference genes across in vivo and in vitro porcine blastocysts, three kinds of programs showed slightly different results; however, there were similar patterns about the rankings of more or less stability overall. In conclusion, SDHA and H2A were determined as the most appropriate reference genes for reliable normalisation in order to find the comparative gene expression in porcine blastocysts produced by different methods, whereas 18S was regarded as a less-stable reference gene. The present study has evaluated the stability of commonly used reference genes for accurate normalisation in porcine embryos to obtain reliable results.

scholarly journals Evaluation of reference genes for gene expression studies in pig muscle tissue by real-time PCR

2011 ◽  
Vol 56 (No. 5) ◽  
pp. 213-216 ◽  
Author(s):  
M. Nesvadbová ◽  
A. Knoll
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Muscle Tissue ◽  
Reference Genes ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
The Stability

The selection of reference genes is essential for gene expression studies when using a real-time quantitative polymerase chain reaction (PCR). Reference gene selection should be performed for each experiment because the gene expression level may be changed in different experimental conditions. In this study, the stability of mRNA expression was determined for seven genes: HPRT1, RPS18, NACA, TBP, TAF4B, RPL32 and OAZ1. The stability of these reference genes was investigated in the skeletal muscle tissue of pig foetuses, piglets and adult pigs using real-time quantitative PCR and SYBR green chemistry. The expression of stability of the used reference genes was calculated using the geNorm application. Different gene expression profiles among the age categories of pigs were found out. RPS18 has been identified as the gene with the most stable expression in the muscle tissue of all pig age categories. HPRT1 and RPL32 were found to have the highest stability in piglets and adult pigs, and in foetuses and adults pigs, respectively. The newly used reference gene, TAF4B, reached the highest expression stability in piglets.

scholarly journals A novel method for visualizing and tracking endogenous mRNA in a specific cell population in pathological neovascularization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Md Imam Uddin ◽  
Tyler C. Kilburn ◽  
Sara Z. Jamal ◽  
Craig L. Duvall ◽  
John S. Penn
Keyword(s):  
Real Time ◽  
Disease Burden ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Fluorescence Emission ◽  
High Sensitivity ◽  
Living Systems ◽  
Specific Cell ◽  
Potential Marker

AbstractDiabetic retinopathy, retinopathy of prematurity and retinal vein occlusion are potentially blinding conditions largely due to their respective neovascular components. The development of real-time in vivo molecular imaging methods, to assess levels of retinal neovascularization (NV), would greatly benefit patients afflicted with these conditions. mRNA hybridization techniques offer a potential method to image retinal NV. The success of these techniques hinges on the selection of a target mRNA whose tissue levels and spatial expression patterns correlate closely with disease burden. Using a model of oxygen-induced retinopathy (OIR), we previously observed dramatic increases in retinal endoglin that localized to neovascular structures (NV), directly correlating with levels of neovascular pathology. Based on these findings, we have investigated Endoglin mRNA as a potential marker for imaging retinal NV in OIR mice. Also of critical importance, is the application of innovative technologies capable of detecting mRNAs in living systems with high sensitivity and specificity. To detect and visualize endoglin mRNA in OIR mice, we have designed and synthesized a novel imaging probe composed of short-hairpin anti-sense (AS) endoglin RNA coupled to a fluorophore and black hole quencher (AS-Eng shRNA). This assembly allows highly sensitive fluorescence emission upon hybridization of the AS-Eng shRNA to cellular endoglin mRNA. The AS-Eng shRNA is further conjugated to a diacyl-lipid (AS-Eng shRNA–lipid referred to as probe). The lipid moiety binds to serum albumin facilitating enhanced systemic circulation of the probe. OIR mice received intraperitoneal injections of AS-Eng shRNA–lipid. Ex vivo imaging of their retinas revealed specific endoglin mRNA dependent fluorescence superimposed on neovascular structures. Room air mice receiving AS-Eng shRNA–lipid and OIR mice receiving a non-sense control probe showed little fluorescence activity. In addition, we found that cells in neovascular lesions labelled with endoglin mRNA dependent fluorescence, co-labelled with the macrophage/microglia-associated marker IBA1. Others have shown that cells expressing macrophage/microglia markers associate with retinal neovascular structures in proportion to disease burden. Hence we propose that our probe may be used to image and to estimate the levels of retinal neovascular disease in real-time in living systems.

scholarly journals Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 960
Author(s):  
Meagan Archer ◽  
Jianping Xu
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Specific Method ◽  
Experimental Conditions ◽  
Reference Gene Selection ◽  
Physiological Processes ◽  
The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

scholarly journals Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca’s response to salinity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Meng Wang ◽  
Tingting Ren ◽  
Prince Marowa ◽  
Haina Du ◽  
Zongchang Xu
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Suitable Reference Gene ◽  
Normalize Gene Expression ◽  
Suaeda Glauca ◽  
Suitable Reference ◽  
Germinating Seeds ◽  
Different Tissues

AbstractQuantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

scholarly journals Optimal use of statistical methods to validate reference gene stability in longitudinal studies

2019 ◽  
Author(s):  
Venkat Krishnan Sundaram ◽  
Nirmal Kumar Sampathkumar ◽  
Charbel Massaad ◽  
Julien Grenier
Keyword(s):  
Statistical Methods ◽  
Reference Gene ◽  
Coefficient Of Variation ◽  
Reference Genes ◽  
Developmental Studies ◽  
Variation Analysis ◽  
Reference Gene Stability ◽  
Statistical Approaches ◽  
The Stability ◽  
Gene Stability

AbstractMultiple statistical approaches have been proposed to validate reference genes in qPCR assays. However, conflicting results from these statistical methods pose a major hurdle in the choice of the best reference genes. Indeed, as their respective approaches to calculating reference gene stability is different, their suitability has to be tested for a given experimental setting. In this study, the stability of 10 candidate reference genes (Actb, Gapdh, Tbp, Sdha, Pgk1, Ppia, Rpl13a, Hsp60, Mrpl10, Rps26) was assessed using four common statistical approaches (GeNorm, NormFinder, Coefficient of Variation analysis and Pairwise ΔCt method) in a longitudinal setting. We used the development of the cerebellum and the spinal cord of mice as a model to assess the suitability of these statistical methods for reference gene validation. GeNorm and the Pairwise ΔCt were found to be ill suited due to a fundamental assumption in their stability calculations. Whereas, NormFinder and Coefficient of Variation analysis fare better provided they are used complementarily. We therefore devised a workflow combining these two methods for validating reference genes in developmental studies. This workflow proves to be more robust than any of the methods used individually.

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