GAPDH, β-actin and β2-microglobulin, as three common reference genes, are not reliable for gene expression studies in equine adipose- and marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2. We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2. In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

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

BioTechniques ◽

10.2144/btn-2019-0098 ◽

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.

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Isogenic-induced endothelial cells enhance osteogenic differentiation of mesenchymal stem cells on silk fibroin scaffold

Regenerative Medicine ◽

10.2217/rme-2018-0166 ◽

2019 ◽

Vol 14 (7) ◽

pp. 647-661 ◽

Cited By ~ 4

Author(s):

Sindhuja D Eswaramoorthy ◽

Nandini Dhiman ◽

Gayathri Korra ◽

Carlo M Oranges ◽

Dirk J Schaefer ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Endothelial Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Silk Fibroin ◽

Expression Studies ◽

Gene Expression Studies ◽

Endothelial Gene Expression

Aim: We investigated the role of induced endothelial cells (iECs) in mesenchymal stem cells (MSCs)/iECs co-culture and assessed their osteogenic ability on silk fibroin nanofiber scaffolds. Methods: The osteogenic differentiation was assessed by the ALP assay, calcium assay and gene expression studies. Results: The osteogenic differentiation of the iECs co-cultures was found to be higher than the MSCs group and proximal to endothelial cells (ECs) co-cultures. Furthermore, the usage of isogenic iECs for co-culture increased the osteogenic and endothelial gene expression. Conclusion: These findings suggest that iECs mimic endothelial cells when co-cultured with MSCs and that one MSCs source can be used to give rise to both MSCs and iECs. The isogenic MSCs/iECs co-culture provides a new option for bone tissue engineering applications.

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Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR

PLoS ONE ◽

10.1371/journal.pone.0046451 ◽

2012 ◽

Vol 7 (9) ◽

pp. e46451 ◽

Cited By ~ 176

Author(s):

Deshui Liu ◽

Lindan Shi ◽

Chenggui Han ◽

Jialin Yu ◽

Dawei Li ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Nicotiana Benthamiana ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Expression Studies ◽

Gene Expression Studies

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Validation of Reference Genes for Studying Different Abiotic Stresses in oat (Avena sativa L.) by RT-qPCR

Plants ◽

10.3390/plants10071272 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1272

Author(s):

Judit Tajti ◽

Magda Pál ◽

Tibor Janda

Keyword(s):

Gene Expression ◽

Avena Sativa ◽

Abiotic Stresses ◽

Reference Genes ◽

Nutritional Value ◽

Tissue Type ◽

Future Research ◽

Experimental Conditions ◽

Expression Studies ◽

Gene Expression Studies

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

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Selection of reference genes for gene expression studies in chronically hypoxic heart

Journal of Molecular and Cellular Cardiology ◽

10.1016/j.yjmcc.2018.05.075 ◽

2018 ◽

Vol 120 ◽

pp. 22-23

Author(s):

D. Benak ◽

D. Sotakova ◽

J. Neckar ◽

F. Kolar ◽

M. Hlavackova

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Hypoxic Heart ◽

Expression Studies ◽

Gene Expression Studies ◽

Selection Of

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Selection of reference genes for measuring the expression of aiiO in Ochrobactrum quorumnocens A44 using RT-qPCR

Scientific Reports ◽

10.1038/s41598-019-49474-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

Dorota M. Krzyżanowska ◽

Anna Supernat ◽

Tomasz Maciąg ◽

Marta Matuszewska ◽

Sylwia Jafra

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Reference Genes ◽

Significance Threshold ◽

Expression Studies ◽

Reverse Transcription Quantitative Pcr ◽

Gene Expression Studies ◽

The Stability ◽

Selection Of

Abstract Reverse transcription quantitative PCR (RT-qPCR), a method of choice for quantification of gene expression changes, requires stably expressed reference genes for normalization of data. So far, no reference genes were established for the Alphaproteobacteria of the genus Ochrobactrum. Here, we determined reference genes for gene expression studies in O. quorumnocens A44. Strain A44 was cultured under 10 different conditions and the stability of expression of 11 candidate genes was evaluated using geNorm, NormFinder and BestKeeper. Most stably expressed genes were found to be rho, gyrB and rpoD. Our results can facilitate the choice of reference genes in the related Ochrobactrum strains. O. quorumnocens A44 is able to inactivate a broad spectrum of N-acyl homoserine lactones (AHLs) – the quorum sensing molecules of many Gram-negative bacteria. This activity is attributed to AiiO hydrolase, yet it remains unclear whether AHLs are the primary substrate of this enzyme. Using the established RT-qPCR setup, we found that the expression of the aiiO gene upon exposure to two AHLs, C6-HLS and 3OC12-HSL, does not change above the 1-fold significance threshold. The implications of this finding are discussed in the light of the role of quorum sensing-interfering enzymes in the host strains.

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Peer Review #1 of "RNA-seq reveals more consistent reference genes for gene expression studies in human non-melanoma skin cancers (v0.1)"

10.7287/peerj.3631v0.1/reviews/1 ◽

2017 ◽

Author(s):

B Davis-Dusenbery

Keyword(s):

Gene Expression ◽

Peer Review ◽

Reference Genes ◽

Rna Seq ◽

Skin Cancers ◽

Expression Studies ◽

Gene Expression Studies ◽

Melanoma Skin

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Selection and Validation of Appropriate Reference Genes for Gene Expression Studies in Schima Superba

10.21203/rs.3.rs-145449/v1 ◽

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.

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