Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Li, Tianyi; Diao, Hong-Ying; Zhao, Lei; Xing, Yue; Zhang, Jichang; Liu, Ning; Yan, Youyou; Tian, Xin; Sun, Weiyi; Liu, Bin

doi:10.1186/s12867-017-0086-z

Cited by 20 publications

(16 citation statements)

References 36 publications

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“…In accordance with previous studies, they identified HPRT1 and YWHAZ as the two most suitable genes in HUVEC-stain treated cells stimulated with TNF- α in relation to gene expression [ 25 ]. However, there was complete discrepancy in two recent studies, which identified TFRC, RPLO, GAPDH, and ACTB as the most stable genes [ 12 ] and the other studies found that hmbs, ywhaz, and tbp were considered as three most stable RGs in MSCs before and after differentiation [ 54 ]. Interestingly, in our study, ywhaz was ranked 1 st , hmbs 3 rd , actb 7 th , tfrc 8 th , gapdh11 th , and rplo 9 th , respectively.…”

Section: Discussionmentioning

confidence: 99%

“…Reverse-transcription-qPCR is one of the most widely used methods directly evolved from the end-point detection PCR to detect gene expression level under different research conditions because of its time-saving, high sensitivity, and specificity [ 12 – 14 ]. But if this technique is performed in an inappropriate way, especially using incorrect housekeeping genes (HKGs), considerable misinterpretation of results will happen [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

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Validation of Housekeeping Genes as Reference for Reverse-Transcription-qPCR Analysis in Busulfan-Injured Microvascular Endothelial Cells

Smith

Sun

et al. 2018

BioMed Research International

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Endothelial cells (ECs) could express some important cytokines and signal molecules which play a key role in normal hematopoiesis and repopulation. Busulfan-induced vascular endothelial injury is an important feature after hematopoietic stem cell transplantation (HSCT). But the molecular mechanism of how the injured ECs affect hematopoietic reconstruction is still unknown. It is possibly through modulation of the change of some gene expression. RT-qPCR is one of the most popular methods used to accurately determine gene expression levels, based on stable reference gene (RG) selection from housekeeping genes. So our aim is to select stable RGs for more accurate measures of mRNA levels during Busulfan-induced vascular endothelial injury. In this study, 14 RGs were selected to investigate their expression stability in ECs during 72 hours of EC injury treated with Busulfan. Our results revealed extreme variation in RG stability compared by five statistical algorithms. ywhaz and alas1 were recognized as the two idlest RGs on account of the final ranking, while the two most usually used RGs (gapdh and actb) were not the most stable RGs. Next, these data were verified by testing signalling pathway genes ctnnb1, robo4, and notch1 based on the above four genes ywha, alas1, gapdh, and actb. It shows that the normalization of mRNA expression data using unstable RGs greatly affects gene fold change, which means the reliability of the biological conclusions is questionable. Based on the best RGs used, we also found that robo4 is significantly overexpressed in Busulfan-impaired ECs. In conclusion, our data reaffirms the importance of RGs selection for the valid analysis of gene expression in Busulfan-impaired ECs. And it also provides very useful guidance and basis for more accurate differential expression gene screening and future expanding biomolecule study of different drugs such as cyclophosphamide and fludarabine-injured ECs.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Validation of Housekeeping Genes as Reference for Reverse-Transcription-qPCR Analysis in Busulfan-Injured Microvascular Endothelial Cells

Smith

Sun

et al. 2018

BioMed Research International

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“…BrightGreen qPCR MasterMix-Low ROX and a Bio-Rad CFX96 were used for performing the qPCR. Primers for TATA-box binding protein (TBP) and Peptidylprolyl isomerase A (PPIA) were used as internal controls as previously described [ 34 ]. ZIKV primers were designed for this work with the following sequences: Forward 5′-CAAAAGGAGGCCCTGGTCAT-3′, Reverse 5′-ATGAAAGACGTCCACCCCAC-3′ (92 bp product).…”

Section: Methodsmentioning

confidence: 99%

Higher Cytopathic Effects of a Zika Virus Brazilian Isolate from Bahia Compared to a Canadian-Imported Thai Strain

Alpuche-Lazcano

McCullogh

Corpo

et al. 2018

Viruses

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Zika virus (ZIKV) is an emerging pathogen from the Flaviviridae family. It represents a significant threat to global health due to its neurological and fetal pathogenesis (including microcephaly and congenital malformations), and its rapid dissemination across Latin America in recent years. The virus has spread from Africa to Asia, the Pacific islands and the Americas with limited knowledge about the pathogenesis associated with infection in recent years. Herein, we compared the ability of the Canadian-imported Thai strain PLCal_ZV and the Brazilian isolate HS-2015-BA-01 from Bahia to produce infectious ZIKV particles and cytopathic effects in a cell proliferation assay. We also compared the intracellular viral RNA accumulation of the two strains by quantitative RT-PCR (reverse transcription polymerase chain reaction) analyses. Our observations show that HS-2015-BA-01 is more cytopathic than PLCal_ZV in proliferation assays in Vero, Human Embryonic Kidney HEK 293T and neuroblastoma SH-SY5Y cells. Quantitative RT-PCR shows that the level of viral RNA is higher with HS-2015-BA-01 than with PLCal_ZV in two cell lines, but similar in a neuroblastoma cell line. The two strains have 13 amino acids polymorphisms and we analyzed their predicted protein secondary structure. The increased cytopathicity and RNA accumulation of the Brazilian ZIKV isolate compared to the Thai isolate could contribute to the increased pathogenicity observed during the Brazilian epidemic.

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“…Their findings regard one cell type with different biological replicates, and with the variability of expression, the ranking by three programs produced different results [58], thus proving that in terms of reference gene identification, using three programs is fully justified. Similarly, in identifying housekeeping genes in HUVECs treated with hydrogen peroxide, RPLP0 was shown to be among the most stable genes, however in our study, this candidate was found to be the least stable gene [59].…”

Section: Analysis Of Candidate Housekeeping Gene Stabilitymentioning

confidence: 43%

Validation of reference genes for real-time PCR of cord blood mononuclear cells, differentiating endothelial progenitor cells, and mature endothelial cells

Royer

Bégin

Laurent

et al. 2018

Experimental Cell Research

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In the last ten years, endothelial progenitor cells (EPCs) have gained interest as an attractive cell population in regenerative medicine for vascular applications. This population is defined as the precursor of endothelial mature cells (ECs) through a process of differentiation. To our knowledge, no single marker can be used to discriminate them from mature ECs. To effectively study their differentiation kinetics, gene expression must be assessed. Quantitative real-time PCR (RT-qPCR) is widely used to analyze gene expression. To minimize the impact of variances from RT-qPCR, a rigorous selection of reference genes must be performed prior to any experiments due to variations in experimental conditions. In this study, CD34+ mononuclear cells were extracted from human cord blood and differentiated into EPCs after seeding for a maximum period of 21 days. To choose the best combinations of reference genes, we compared the results of EPCs, CD34+ mononuclear cells, and mature endothelial cells to ensure that the differentiation kinetics did not affect the expression of our selected reference genes. The expression levels of seven genes, namely, YWHAZ, GAPDH, HPRT1, RPLP0, UBC, B2M, and TBP were thus compared. The algorithms geNorm, NormFinder, BestKeeper, and the Comparative ΔCt method were employed to assess the expression of each candidate gene. Overall results reveal that the expression stability of reference genes may differ depending on the statistical program used. YWHAZ, GAPDH, and UBC composed the optimal set of reference genes for the gene expression studies performed by RT-qPCR in our experimental conditions. This work can thus serve as a starting point for the selection of candidate reference genes to normalize the levels of gene expression in endothelial progenitor cell populations.

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Identification of suitable reference genes for real-time quantitative PCR analysis of hydrogen peroxide-treated human umbilical vein endothelial cells

Cited by 20 publications

References 36 publications

Validation of Housekeeping Genes as Reference for Reverse-Transcription-qPCR Analysis in Busulfan-Injured Microvascular Endothelial Cells

Validation of Housekeeping Genes as Reference for Reverse-Transcription-qPCR Analysis in Busulfan-Injured Microvascular Endothelial Cells

Higher Cytopathic Effects of a Zika Virus Brazilian Isolate from Bahia Compared to a Canadian-Imported Thai Strain

Validation of reference genes for real-time PCR of cord blood mononuclear cells, differentiating endothelial progenitor cells, and mature endothelial cells

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