scholarly journals Selection and validation of reference genes for real-time qRT-PCR normalization in different tissues of Eucalyptus tereticornis

2012 ◽  
Vol 61 (1-6) ◽  
pp. 280-286
Author(s):  
B. Karpaga Raja Sundari ◽  
M. Ghosh Dasgupta
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Genomic Research ◽  
Initiation Factor ◽  
Primary Cell Wall ◽  
Stable Gene ◽  
Eucalyptus Tereticornis ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Different Tissues

AbstractReference genes are generally used as endogenous normalization factor for relative quantification of target genes in quantitative real-time PCR (qRT-PCR). The present work aimed at identifying suitable reference genes for normalization of qRT-PCR data in tissues of Eucalyptus tereticornis. The expression levels of housekeeping genes like Actin (EtAct2), Isocitrate dehy - drogenase (EtIDH), ribosomal RNA (Et18s rRNA), SAND family protein (EtSAND), Histone protein (EtH2B), α-Tubulin (EtTUB), and eukaryotic initiation factor (EteIF4B) were studied to characterize their normalization stability in different tissues including young leaves, internodes, developing and mature xylem. The expression level of these genes was analyzed using different algorithms like geNorm, NormFinder and Best- Keeper. Among the seven reference genes analyzed, EtAct2 was expressed with less variance and was found to be the most stable reference gene across different tissues using all the three programs, while the least stable gene identified was EtH2B. Further, the normalization efficiency of the reference genes were assessed to predict the expression levels of three primary cell wall specific cellulose synthase transcripts (EtCesAs) in E. tereticornis tissues. The relative expression of EtCesA4, EtCesA5 and EtCesA6 was determined to be 3-19 fold higher in leaf and internode tissues when compared to developing and mature xylem tissues. This study will allow accurate normalization of qRT-PCR experiments across different tissues in E. tereticornis for future genomic research in this tropical eucalypt species.

scholarly journals Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Forests ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 193
Author(s):  
Dandan Li ◽  
Sen Yu ◽  
Minzhen Zeng ◽  
Xiao Liu ◽  
Jia Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Real Time ◽  
Reference Genes ◽  
Pcr Analysis ◽  
Stable Gene ◽  
Larix Olgensis ◽  
Qrt Pcr ◽  
Study Gene Expression ◽  
Selection Of

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

scholarly journals Identification of stable reference genes for lipopolysaccharide-stimulated macrophage gene expression studies

2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Roshini Kalagara ◽  
Weimin Gao ◽  
Honor L. Glenn ◽  
Colleen Ziegler ◽  
Laura Belmont ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Reference Gene ◽  
Reference Genes ◽  
Target Genes ◽  
Stable Gene ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Expression Studies ◽  
Gene Expression Studies

Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated macrophages to model immune signaling are widely used for elucidating the mechanisms of inflammation-related disease. When expression levels of target genes are quantified using Real-Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference genes, which should have stable expression. Judicious selection of reference genes is, therefore, critical to interpretation of qRT-PCR results. Ideal reference genes must be identified for each experimental system and demonstrated to remain constant under the experimental conditions. In this study, we evaluated the stability of eight common reference genes: Beta-2-microglobulin (B2M), Cyclophilin A/Peptidylprolyl isomerase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), Hypoxanthine Phosphoribosyltransferase 1, Large Ribosomal Protein P0, TATA box binding protein, Ubiquitin C (UBC), and Ribosomal protein L13A. Expression stability of each gene was tested under different conditions of LPS stimulation and compared to untreated controls. Reference gene stabilities were analyzed using Ct value comparison, NormFinder, and geNorm. We found that UBC, closely followed by B2M, is the most stable gene, while the commonly used reference gene GAPDH is the least stable. Thus, for improved accuracy in evaluating gene expression levels, we propose the use of UBC to normalize PCR data from LPS-stimulated macrophages.

scholarly journals Selection of reference genes for quantitative real-time PCR analysis in cucumber (Cucumis sativus L.), pumpkin (Cucurbita moschata Duch.) and cucumber–pumpkin grafted plants

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6536 ◽  
Author(s):  
Li Miao ◽  
Xing Qin ◽  
Lihong Gao ◽  
Qing Li ◽  
Shuzhen Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Stable Expression ◽  
Quantitative Real Time Pcr ◽  
Graft Union ◽  
Expression Levels ◽  
Qrt Pcr

Background Quantitative real-time PCR (qRT-PCR) is a commonly used high-throughput technique to measure mRNA transcript levels. The accuracy of this evaluation of gene expression depends on the use of optimal reference genes. Cucumber–pumpkin grafted plants, made by grafting a cucumber scion onto pumpkin rootstock, are superior to either parent plant, as grafting conveys many advantages. However, although many reliable reference genes have been identified in both cucumber and pumpkin, none have been obtained for cucumber–pumpkin grafted plants. Methods In this work, 12 candidate reference genes, including eight traditional genes and four novel genes identified from our transcriptome data, were selected to assess their expression stability. Their expression levels in 25 samples, including three cucumber and three pumpkin samples from different organs, and 19 cucumber–pumpkin grafted samples from different organs, conditions, and varieties, were analyzed by qRT-PCR, and the stability of their expression was assessed by the comparative ΔCt method, geNorm, NormFinder, BestKeeper, and RefFinder. Results The results showed that the most suitable reference gene varied dependent on the organs, conditions, and varieties. CACS and 40SRPS8 were the most stable reference genes for all samples in our research. TIP41 and CACS showed the most stable expression in different cucumber organs, TIP41 and PP2A were the optimal reference genes in pumpkin organs, and CACS and 40SRPS8 were the most stable genes in all grafted cucumber samples. However, the optimal reference gene varied under different conditions. CACS and 40SRPS8 were the best combination of genes in different organs of cucumber–pumpkin grafted plants, TUA and RPL36Aa were the most stable in the graft union under cold stress, LEA26 and ARF showed the most stable expression in the graft union during the healing process, and TIP41 and PP2A were the most stable across different varieties of cucumber–pumpkin grafted plants. The use of LEA26, ARF and LEA26+ARF as reference genes were further verified by analyzing the expression levels of csaCYCD3;1, csaRUL, cmoRUL, and cmoPIN in the graft union at different time points after grafting. Discussion This work is the first report of appropriate reference genes in grafted cucumber plants and provides useful information for the study of gene expression and molecular mechanisms in cucumber–pumpkin grafted plants.

geNorm assessment of the efficacy of novel reference genes ALG9, TFCP2 and ZNF410 compared to GAPDH for real time normalization in GI carcinoids

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20052-20052
Author(s):  
G. Eick ◽  
M. Kidd ◽  
S. Mane ◽  
B. Nadler ◽  
M. Champaneria ◽  
...  
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Large Scale ◽  
Target Genes ◽  
Marker Genes ◽  
Stable Gene ◽  
Rt Pcr ◽  
Experimental Conditions ◽  
Expression Levels ◽  
Accurate Normalization

20052 Background: Robust quantitation of potential clinical marker genes using quantitative real-time PCR (Q RT-PCR) is critically dependent on accurate normalization. Although GAPDH has historically been used for normalization, its expression has been shown to vary widely between different tissues and experimental conditions. Additionally, conventional normalization strategies based on a single housekeeping gene can lead to large normalization errors. The determination of a panel of genes that have robust expression in the experimental system being studied is therefore essential to ensure accurate normalization and interpretation of results. Methods: Based upon the availability of large-scale gene databases, we developed methodology to identify highly expressed genes (mean log-transformed expression levels: 4–8 in all samples) with low variability (S.D. < 0.22) in a Affymetrix U133A dataset of 36 gastrointestinal tumors and normal tissues. Eight novel candidate reference genes were identified and their expression levels established by Q RT-PCR in an independent set of GI tissue samples (n = 24). The geNorm tool was used to identify the most stably expressed set of genes amongst the 8 candidate genes. The expression levels of 3 potential GI tumor marker genes, namely the adhesin MAGE-D2, the metastasis-associated MTA1, and the mitosis regulator, NAP1L1, were normalized to GAPDH or geNorm and compared. Results: geNorm identified 3 genes, ALG9, TFCP2 and ZNF410, as the most robustly expressed control genes. GAPDH, in contrast, exhibited the highest variability and was considered the least stable gene of the nine evaluated. Two previously-identified target genes, MAGE-D2 and MTA1, were significantly elevated (p < 0.05) in malignant tumor samples (vs normal GI samples) when normalized by geNormATZ but not when normalized using GAPDH. NAP1L1 was only over-expressed in small intestinal carcinoids (normalized to geNormATZ). Expression levels of this gene were high in normal gastric mucosa. Conclusions: We provide a robust basis for the establishment of a reference gene set using GeneChip data and provide evidence for the clinical utility of identifying reference genes using the geNorm approach in GI neuroendocrine tumors. No significant financial relationships to disclose.

scholarly journals Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover (Trifolium repens L.) Involved in Five Abiotic Stresses

Plants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 996
Author(s):  
Qi Pu ◽  
Zhou Li ◽  
Gang Nie ◽  
Jiqiong Zhou ◽  
Lin Liu ◽  
...  
Keyword(s):  
Real Time ◽  
White Clover ◽  
Trifolium Repens ◽  
Abiotic Stresses ◽  
Reference Genes ◽  
Grass Species ◽  
Stable Gene ◽  
Experimental Conditions ◽  
Qrt Pcr ◽  
Trifolium Repens L

White clover (Trifolium repens L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC, and ABC) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that 60SrRNA and ACT101 were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, SAMDC, UBQ, 60SrRNA, and ACT101 were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of Cyt-Cu/Zn SOD and CAT confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.

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 Cloning and evaluation of reference genes for quantitative real-time PCR analysis inAmorphophallus

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3260 ◽  
Author(s):  
Kai Wang ◽  
Yi Niu ◽  
Qijun Wang ◽  
Haili Liu ◽  
Yi Jin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Real Time ◽  
Reference Genes ◽  
Dynamic Range ◽  
Expression Profiles ◽  
Mrna Levels ◽  
Expression Stability ◽  
Degenerate Primers ◽  
Different Tissues

Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes.Amorphophallusis a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes inAmorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genesAmorphophalluswere cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) inA. albusandA. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated thatEF1-a,EIF4A,H3andUBQwere the best reference genes under heat stress inAmorphophallus. Furthermore,EF1-a,EIF4A,TUB, andRPwere the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined thatEF1-α,EIF4A,andCYPwere stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock proteinSHSP, which is related to heat stress inAmorphophallus. In sum,EF1-αandEIF4Awere the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR inAmorphophallus.

scholarly journals Selection and validation of reference genes for quantitative gene expression analyses in persimmon (Diospyros kaki Thunb.) using real-time quantitative PCR

2019 ◽  
Vol 70 (4) ◽  
pp. 261-267
Author(s):  
Gaigai Du ◽  
Liyuan Wang ◽  
Huawei Li ◽  
Peng Sun ◽  
Jianmin Fu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Developmental Stages ◽  
Suitable Reference Gene ◽  
Diospyros Kaki ◽  
Stable Gene ◽  
Expression Studies ◽  
Gene Expression Studies

Background and aims Persimmon (Diospyros kaki) is an economically important fruit tree species with complex flowering characteristics. To obtain accurate expression pattern analysis results, it is vital to select a reliable gene for the normalization of real-time quantitative polymerase chain reaction data. The aim of this study was to identify the optimal internal control gene among six candidate genes for gene expression analysis in different persimmon organs and developmental stages. Materials and methods This analysis was conducted using geNorm and NormFinder software to show differences in the stability of the six reference genes among tissues and floral developmental stages of the same plant. Results Although genes that exhibited moderate expression in NormFinder revealed slightly different expression stabilities than those obtained by geNorm, both sets of results showed that GAPDH was the best reference gene in different organs and floral buds at different developmental stages, whereas 18SrRNA was the least stable gene. Conclusions Based on the overall ranking, GAPDH is the most suitable reference gene and is highly recommended for gene expression studies in different organs and different developmental stages of persimmon. This study provides useful reference data for future gene expression studies and will contribute to improving the accuracy of gene expression results in persimmon.

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 Identification of miR-328-3p as an endogenous reference gene for the normalization of miRNA expression data from patients with Diabetic Retinopathy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marcelle SanJuan Ganem Prado ◽  
Thaline Cunha de Goes ◽  
Mirthz Lemos de Jesus ◽  
Lucilla Silva Oliveira Mendonça ◽  
Jadson Santos Nascimento ◽  
...  
Keyword(s):  
Diabetic Retinopathy ◽  
Mirna Expression ◽  
Reference Genes ◽  
Data Interpretation ◽  
Stable Gene ◽  
Internal Reference ◽  
Expression Levels ◽  
Rna Molecules ◽  
Gene Normalization ◽  
Mirna Expression Data

AbstractDiabetic Retinopathy, the main cause of visual loss and blindness among working population, is a complication of Diabetes mellitus (DM), which has been described as a major public health challenge, so it is important to identify biomarkers to predict and to stratify patient´s possibility for developing DR. MicroRNAs (miRNAs) are small non-coding RNA molecules that have showed to be promising disease biomarkers and association of miRNAs with the possibility to develop DR has been reported. However, evaluating miRNA expression involves normalization of RT-qPCR data using internal reference genes that should be properly determined, considering their impact on expression levels calculation and, until date, there is no unanimity on reference miRNAs for the investigation of circulating miRNAs in DR. We aimed to estimate the appropriateness of a group of miRNAs as normalizers to identify which might be considered steady internal reference genes in expression studies on DR plasma samples. Expression levels of candidates were analyzed in 60 healthy controls, 48 DM without DR patients and 62 DR patients with two statistical tools: NormFinder and RefFinder. MiR-328-3p was the most stable gene and we also investigated the effect of gene normalization, demonstrating that different normalization strategies have important implications for accurate data interpretation.

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