scholarly journals Identification and validation of suitable reference genes for quantitative real-time PCR gene expression analysis in pregnant human myometrium

2021 ◽  
Author(s):  
Sarah Arrowsmith
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Geometric Mean ◽  
Optimal Number ◽  
Pairwise Variation ◽  
Human Myometrium ◽  
Relative Expression ◽  
Accurate Normalisation ◽  
Suitable Reference ◽  
Accurate Quantification

AbstractAccurate quantification of quantitative PCR (qPCR) data requires a set of stable reference genes (RGs) for normalisation. Despite its importance to mechanistic studies, no evaluation of RG stability has been conducted for pregnant human myometrium. A systematic search of the literature was performed to identify the most used RGs in human myometrial gene expression studies. The stability of these genes, and others, was then evaluated using geNorm and NormFinder algorithms, in samples of myometrium from singleton or twin pregnancies (n = 7 per group) delivering at term or preterm. The most frequently cited RGs were GAPDH, ACTB, B2M and 18s. There was strong agreement between algorithms on the most and least stable genes: Both indicated CYC1, YWHAZ and ATP5B were the most stably expressed. Despite being some of the most used RGs, B2M, 18s and ACTB expression was least stable and was too variable for use as accurate normalisation factors. Pairwise variation analysis determined that the optimal number of RGs for accurate normalisation is two. Validation of the choice of RGs by comparing relative expression of oxytocin receptors (OXTR) using the least stable 18s and B2M, with the most stable, CYC1 and YWHAZ, erroneously demonstrated significantly increased OXTR expression in myometrium in singleton pregnancies compared to twins. This study demonstrates the importance of appropriate RG selection for accurate quantification of relative expression in pregnant human myometrium qPCR studies. For normalisation, the geometric mean of CYC1 and YWHAZ or ATP5B is suggested. The use of ACTB, 18s and B2M, is not recommended.

scholarly journals Selection of reference genes suitable for normalization of RT-qPCR data in glioma stem cells

BioTechniques ◽  
2020 ◽  
Vol 68 (3) ◽  
pp. 130-137
Author(s):  
Weiqi Dang ◽  
Xiang Zhang ◽  
Qinghua Ma ◽  
Lu Chen ◽  
Mianfu Cao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Reference Genes ◽  
Geometric Mean ◽  
Specific Reference ◽  
Glioma Stem Cells ◽  
Reverse Transcription Pcr ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Suitable Reference

Considering the importance of gene expression studies for understanding the biology of glioma stem cells (GSCs), we aimed to identify the reliable reference genes in GSCs that were derived from the glioma cell lines T98G, LN229, 090116 and 091214. Quantitative real-time reverse-transcription PCR was employed using 11 reference genes identified through a PubMed literature search, and the assessment of stability through the geNorm, Normfinder and coefficient of variation methods was performed to select suitable reference genes. We found that HPRT1 and RPL13A were the most suitable reference genes, and validated the geometric mean of these genes to normalize the expression of stemness genes by GSCs. Therefore, it is necessary to select novel cell-specific reference genes with greater expression stability for gene expression studies in GSCs.

scholarly journals Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Won-Jae Lee ◽  
Ryoung-Hoon Jeon ◽  
Si-Jung Jang ◽  
Ji-Sung Park ◽  
Seung-Chan Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Optimal Number ◽  
Experimental Conditions ◽  
Pearson’S Correlation ◽  
Qrt Pcr ◽  
Differentiated Cells ◽  
Pearson's Correlation ◽  
Suitable Reference

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson’s correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson’s correlation revealed high correlation (r>0.9) between NF3and NFopt. Overall, the present study showed thatHMBS,YWHAZ,SDHA, andTBPare suitable reference genes for qRT-PCR in porcine MSCs.

scholarly journals Normalization Strategy for Selection of Reference Genes for RT-qPCR Analysis in Left Ventricles of Failing Human Hearts

2021 ◽  
Author(s):  
Zdenko Červenák ◽  
Filip Červenák ◽  
Adriana Adamičková ◽  
Barbara Šalingová ◽  
Andrea Gažová ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Potential Role ◽  
Geometric Mean ◽  
Housekeeping Genes ◽  
Cq Method ◽  
Differential Gene ◽  
Suitable Reference ◽  
Selection Of

Abstract Introduction: Quantitative RT-PCR is a valuable tool for assessing the gene expression in different human tissues, particularly due to its exceptional sensitivity, accuracy and reliability. However, the choice of adequate control for normalization is a crucial step, greatly affecting the results of all subsequent analyses. So far, only a few studies were focused on the selection of optimal reference genes in left ventricles of failing human hearts, leading to several disparities in experimental results focused on differential gene expression in this area. Therefore, the main objective of this study was to identify a set of suitable reference genes in normal and failing left ventricle tissues, which could increase the reliability of RT-qPCR-based studies in the future. Methods: We analyzed the expression of 15 commonly used housekeeping genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC and YWHAZ) in left ventricles of normal and failed hearts with two-step approach. In the first step, we excluded genes which are variantly expressed using ANOVA-based statistical method. Afterwards, the remaining genes were analyzed using geNorm, NormFinder and BestKeeper algorithms, together with comparative delta Cq method. Finally, the geometric mean of gene rankings across all five methods was calculated.Results: Our analysis identified IPO8, PGK1 and POLR2A as the most stably expressed genes, whereas ACB2 and B2M were found to be expressed variantly, suggesting a potential role of these genes in the pathophysiological processes in failing human hearts.Discussion/Conclusion: Using our two-step approach, we identified and validated three reference genes expressed invariantly in left ventricles of both healthy and failing human hearts, as well as provided a guideline for the selection of reference genes in studies comparing gene expression in these types of tissues.

scholarly journals Identification of reference genes for real-time PCR gene expression studies during seed development and under abiotic stresses in Cyamopsis tetragonoloba (L.) Taub

2018 ◽  
Author(s):  
Poonam Subhash Jaiswal ◽  
Navneet Kaur ◽  
Gursharn Singh Randhawa
Keyword(s):  
Gene Expression ◽  
Seed Development ◽  
Reference Genes ◽  
Suitable Reference Gene ◽  
Cyamopsis Tetragonoloba ◽  
Expression Data ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Suitable Reference ◽  
Accurate Quantification

AbstractGuar (Cyamopsis tetragonoloba) is an important industrial crop. The knowledge about genes of guar involved in various processes can help in developing improved varieties of this crop. qRT-PCR is a preferred technique for accurate quantification of gene expression data. This technique requires the use of appropriate reference genes from the crop to be studied. Such genes have not been yet identified in guar. The expression stabilities of the 10 candidate reference genes, viz., CYP, ACT 11, EF-1α, TUA, TUB, ACT 7, UBQ 10, UBC 2, GAPDH and 18S rRNA were evaluated in various tissues of guar under normal and abiotic stress conditions. Four different algorithms, geNorm, NormFinder, BestKeeper and ∆Ct approach, were used to assess the expression stabilities and the results obtained were integrated into comprehensive stability rankings. The most stable reference genes were found to be CYP and ACT 11(tissues), ACT 11, UBC 2 and ACT 7 (seed development), ACT 7 and TUB (drought stress), TUA, UBC 2 and CYP (nitrogen stress), TUA and UBC 2 (cold stress), GAPDH and ACT 7 (heat stress) and GAPDH and EF-1a (salt stress). These results indicated the necessity of identifying a suitable reference gene for each experimental condition. Four selected reference genes were validated by normalizing the expression of CtMT1 gene. To the best of our knowledge this is the first report on the identification of reference genes in guar. These findings are likely to provide a boost to the gene expression studies in this important crop.

scholarly journals Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tingting Li ◽  
Weigao Yuan ◽  
Shuai Qiu ◽  
Jisen Shi
Keyword(s):  
Gene Expression ◽  
Somatic Embryogenesis ◽  
Reference Genes ◽  
Gene Selection ◽  
Elongation Factor ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Suitable Reference ◽  
Functional Analyses ◽  
Elongation Factor 1

AbstractThe differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant 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 Selection and Validation of Appropriate Reference Genes for Gene Expression Studies in Schima Superba

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.

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 Selecting Suitable Reference Genes for qPCR Normalization: A Comprehensive Analysis in MCF-7 Breast Cancer Cell Line

2020 ◽  
Author(s):  
Nityanand Jain ◽  
Dina Nitisa ◽  
Valdis Pirsko ◽  
Inese Cakstina
Keyword(s):  
Gene Expression ◽  
Cell Line ◽  
Reference Gene ◽  
Reference Genes ◽  
Cancer Cell Line ◽  
Internal Controls ◽  
Reference Gene Expression ◽  
Qpcr Normalization ◽  
Suitable Reference ◽  
Mcf 7

Abstract BackgroundMCF-7 breast cancer cell line is undoubtedly amongst the most extensively studied patient-derived research models, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, have previously identified suitable reference genes for qPCR normalization in MCF-7 cell line. However, over the course of identification of suitable reference genes, a comparative analysis comprising these genes together in a single study have not been reported. Furthermore, the expression dynamics of these reference genes within sub-clones cultured over multiple passages (p) has attracted limited attention from research groups. Therefore, we investigated the expression dynamics of 12 previously suggested reference genes within two sub-clones (culture A1 and A2) cultured identically over multiple passages. Additionally, the effect of nutrient stress on reference gene expression was examined to devise an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies.ResultsThe analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as the least variable reference gene pair while for culture A2, GAPDH-RNA28S was identified. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization of AURKA and KRT19 with triplet pair GAPDH-PCBP1-CCSER2 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress.ConclusionsThe variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-PCBP1-CCSER2 triplet offers a reliable alternative to otherwise traditionally used internal controls for optimizing intra- and inter-assay gene expression differences. Furthermore, we suggest avoiding the use of ACTB, GAPDH and PGK1 as single internal controls.

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