UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines

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.

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High-Dose Edible Bird’s Nest Extract (EBN) Upregulates LDL-R via Suppression of HMGCR Gene Expression in HEPG2 Cell Lines

Sains Malaysiana ◽

10.17576/jsm-2020-4910-09 ◽

2020 ◽

Vol 49 (10) ◽

pp. 2433-2442

Author(s):

Mohd Noor Akmal ◽

Intan-Shameha Abdul Razak ◽

Rozaihan Mansor ◽

Aini Ideris ◽

Abdul Rahman Omar ◽

...

Keyword(s):

Gene Expression ◽

Hepg2 Cell ◽

Cell Lines ◽

High Dose ◽

Edible Bird’S Nest ◽

Hepg2 Cell Lines

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

Scientific Reports ◽

10.1038/s41598-021-84518-w ◽

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.

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

Scientific Reports ◽

10.1038/s41598-021-88151-5 ◽

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.

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

10.7287/peerj.preprints.27695 ◽

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.

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

10.21203/rs.3.rs-42293/v2 ◽

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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Transcriptomic analysis of marine endophytic fungi extract identifies highly enriched anti-fungal fractions targeting cancer pathways in HepG2 cell lines

BMC Genomics ◽

10.1186/s12864-020-6684-z ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Ethel Juliet Blessie ◽

Wasco Wruck ◽

Benaiah Annertey Abbey ◽

Audrey Ncube ◽

Nina Graffmann ◽

...

Keyword(s):

Hepg2 Cell ◽

Cell Lines ◽

Endophytic Fungi ◽

Transcriptomic Analysis ◽

Cancer Pathways ◽

Hepg2 Cell Lines ◽

Anti Fungal

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Green Synthesis of Silver Nanocomposites of Nigella sativa Seeds Extract for Hepatocellular Carcinoma

Current Nanomaterials ◽

10.2174/2468187309666190906130115 ◽

2019 ◽

Vol 4 (3) ◽

pp. 191-200 ◽

Cited By ~ 1

Author(s):

Afreen Usmani ◽

Anuradha Mishra ◽

Asif Jafri ◽

Md Arshad ◽

Mohd Aftab Siddiqui

Keyword(s):

Hepatocellular Carcinoma ◽

Green Synthesis ◽

Hepg2 Cell ◽

Cell Lines ◽

Nigella Sativa ◽

Uv Spectroscopy ◽

Silver Nanocomposites ◽

Natural Drugs ◽

Hepg2 Cell Lines

Background: Silver nanoparticles play a significant role in bioavailability and refining the compatibility of natural drugs in the treatment of various chronic diseases including different types of cancer. Objective: Green synthesis of silver nanocomposites of Nigella sativa seeds extract to evaluate the anticancer effects against hepatocellular carcinoma using HepG2 cell lines. Methods: The AgNCs were developed by treating aqueous extract of N. sativa seeds treated with silver nitrate (1mM) solution and were used to test its efficacy against hepatocellular carcinoma using HepG2 cell lines. Results and Discussion: The Surface Plasmon Resonance (SPR) of prepared AgNCs showed a peak at 432 nm via UV spectroscopy. The selected N. sativa AgNCs were characterized for polydispersity, surface charge and size and the results showed 0.215±0.093 polydispersity index (PDI), zeta potential 18.8±0.372 mV and size range 10-20 nm, respectively. The Fourier transform infrared spectroscopy (FTIR) also showed various peak of functional groups that are possibly involved in the reduction of silver ion and synthesized the N. sativa silver nanocomposites, respectively. N. sativa AgNCs showed 89.954% drug release while in the case of extract release, it was only 33.821% in 24 hrs. Further, in vitro studies of N. sativa AgNCs against hepatocellular carcinoma showed good cytotoxic effect p<0.05 with 7.16 µg/ml IC50 value. Conclusion: Thus, the present results revealed that green synthesis of N. sativa AgNCs can be an alternative tool for clinical application in cancer therapy; however, there is a need to find the mechanism and role of AgNCs inside the individual.

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