Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

Ledderose, Carola; Heyn, Jens; Limbeck, Elisabeth; Kreth, Simone

doi:10.1186/1756-0500-4-427

Cited by 105 publications

(91 citation statements)

References 28 publications

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“…More reliable qPCR data normalization should be achieved using a combination of genes from different biological pathways, rather than a single gene. Our selection of reference genes for bovine neutrophils differs from results obtained for human neutrophils whereby recommended reference genes included TBP, ACTB, and SDHA [11] or GNB2L1, HPRT1, RPL32, ACTB, and B2M [10], and also differs from results we obtained for sheep neutrophils, whereby SDHA|G6PD and GAPDH|YWHAZ were the best pairs of references genes [13]. In lactating dairy cows, YWHAZ along with SDHA and 18S rRNA, were considered the most suitable reference genes for polymorphonuclear leukocytes [14].…”

Section: Stability Of Neutrophil Reference Genes Evaluated In This Studycontrasting

confidence: 51%

“…Eleven genes were selected from commonly used reference genes [11]: ACTB, B2M, G6PD, GAPDH, GYPC, HPRT, PGK1, RPL19, SDHA, TFRC, and YWHAZ. The length of the primers ranged from 18-bp to 27-bp, GC content varied from 45% to 60%, and the expected PCR product size was 71-bp to 126-bp ( Table 2).…”

Section: Candidate Genes For Expression Studiesmentioning

confidence: 99%

“…Neutrophils use two general mechanisms to kill microorganisms: extracellular killing via neutrophil extracellular traps [7][8][9], or intracellular killing via phagocytosis followed by the secretion of antimicrobial proteins, proteolytic enzymes, and reactive oxygen species when membrane-bound granules fuse with phagocytic vesicles. We and others have reported on potential reference genes in human [10,11] and ovine neutrophils [12,13]. In one study with human neutrophils, suitable reference genes included a TATA box binding protein (TBP), beta-actin (ACTB), and succinate dehydrogenase complex subunit A (SDHA) [11].…”

Section: Introductionmentioning

confidence: 99%

“…We and others have reported on potential reference genes in human [10,11] and ovine neutrophils [12,13]. In one study with human neutrophils, suitable reference genes included a TATA box binding protein (TBP), beta-actin (ACTB), and succinate dehydrogenase complex subunit A (SDHA) [11]. In another human study, guanine nucleotide binding protein, ß-peptide 2-like1 (GNB2L1), hypoxanthine phosphoribosyl-transferase 1 (HPRT), ribosomal protein L32 (RPL32), ACTB, and beta-2-microglobulin (B2M) were suggested as suitable reference genes in gene expression studies of neutrophils [10].…”

Section: Introductionmentioning

confidence: 99%

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Reference gene selection for quantitative PCR studies in bovine neutrophils

Vorachek¹,

Bobe²,

Hall³

2013

ABB

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Reference genes are essential for studying mRNA expression with quantitative PCR (qPCR). We investigated 11 candidate whole-blood neutrophil reference genes (ACTB, B2M, G6PD, GAPDH, GYPC, HPRT, PGK1, RPL19, SDHA, TFRC, and YWHAZ) for beef calves, both males and females, with or without selenium supplementation. Initial screening was based on gene expression level (<28 Cq cycles), variability (SD < 1.5 Cq cycles), excluded GYPC and TFRC from further analysis. Expression stability of the remaining genes was evaluated using four software programs: geNorm, NormFinder, BestKeeper, and the comparative delta Cq method. The neutrophil reference genes, YWHAZ, PGK1, and RPL19, consistently ranked among the top four most stable genes under these experimental conditions. The commonly used reference genes, ACTB and HPRT, were not reliable, underscoring the need to validate neutrophil reference genes under different experimental conditions. Multiple reference genes rather than a single gene may provide more robust and reliable results. The best pair of reference genes in whole-blood neutrophils from beef calves overall was PGK1|YWHAZ.

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Section: Stability Of Neutrophil Reference Genes Evaluated In This Studycontrasting

confidence: 51%

Section: Candidate Genes For Expression Studiesmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Reference gene selection for quantitative PCR studies in bovine neutrophils

Vorachek¹,

Bobe²,

Hall³

2013

ABB

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“…The cellular gene IPO8 was quantified as an internal standard for cellular RNA recovery by using forward primer 5′-GCTCTGATAACTGTGCAG-3′, reverse primer 5′-CAGTGTGTACACCTCCTG-3′, and probe 5′-[6FAM]TGCTGTCCTCTGAT-CCTCGC[TAMRA]-3′. IPO8 is stably expressed in both rCD4 and activated T cells (33). This assay has the ability to detect one cell producing HIV-1 RNA in a background of 2 million HIV-1 negative cells and has a limit of quantification of three copies.…”

Section: Methodsmentioning

confidence: 99%

Quantification of HIV-1 latency reversal in resting CD4 ⁺ T cells from patients on suppressive antiretroviral therapy

Cillo

Sobolewski

Bosch

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

213

218

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Reversal of proviral latency is being pursued as a curative strategy for HIV-1 infection. Recent clinical studies of in vivo administration of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) show increases in unspliced cellular HIV-1 RNA levels in resting CD4 + T cells. A critical unknown, however, is the proportion of latent proviruses that can be transcriptionally reactivated by SAHA or T-cell activation. In this study, we quantified the fraction of HIV-1 proviruses in resting CD4 + T cells from patients on suppressive antiretroviral therapy that were reactivated ex vivo with SAHA or antibodies to CD3/CD28. At concentrations of SAHA achieved clinically, only 0.079% of proviruses in resting CD4 + T cells were reactivated to produce virions, compared with 1.5% of proviruses in cells treated with anti-CD3/CD28 antibodies after correcting for spontaneous virion production in the medium control. A significant positive correlation (ρ = 0.67, P < 0.001) was found between levels of virions in the supernatant and unspliced cellular HIV-1 RNA following anti-CD3/CD28 treatment, but not following SAHA treatment (ρ = 0.21, P = 0.99). These results reveal that the majority of HIV-1 proviruses are not reactivated by current therapeutic approaches and that more effective means of reversing proviral latency will likely be required to deplete HIV-1 reservoirs.HIV-1 persistence | HIV-1 eradication | HIV-1 cure | fractional provirus expression A ntiretroviral therapy (ART) for HIV-1 infection suppresses viral replication but is not curative. Assays of infectious virus recovery from quiescent CD4 + T cells isolated from patients on ART have revealed the existence of a reservoir of latent, replication competent HIV-1 with a half-life of 44 mo (1-4). In addition, low-level plasma viremia persists indefinitely on ART (5, 6), and the level of virus in plasma rebounds following cessation of ART (7,8). New therapeutic approaches are required to eliminate both persistent low-level viremia and the latent proviral reservoir. A "kick and kill" approach has been proposed in which latency reversing agents, administered in conjunction with ART, will "kick" proviruses out of latency, followed by a "kill" of the infected cells through viral cytopathic effects or immune-mediated cytotoxicity.Histone deacetylase inhibitors (HDACi) have been proposed as latency reversing agents, and single-dose or multidose administration of suberoylanilide hydroxamic acid (SAHA; vorinostat) in vivo was shown to increase expression of unspliced cellular HIV-1 RNA in resting CD4 + T (rCD4) cells in patients on suppressive ART (9, 10). Although three-to fivefold increases in cellular HIV-1 RNA were observed (9), the fraction of latent HIV-1 proviruses that were reactivated by SAHA was not quantified. It is possible that SAHA transcriptionally reactivated many latent proviruses, or alternatively reactivated only a minority of latent proviruses. These two alternatives have very different implications in terms of the impact SAHA coul...

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Single‐Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry

et al. 2020

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Understanding how immune cells respond to external stimuli such as pathogens or drugs is a key component of biomedical research. Critical to the immune response are the expression of cell‐surface receptors and the secretion of cytokines, which are tightly regulated by gene expression and protein synthesis. Previously, cytokine mRNA expression levels have been measured from bulk analysis of heterogeneous or sorted cell populations, and the correlation between cytokine mRNA expression and protein levels using these techniques can be highly variable. Flow cytometry is used to monitor changes in cell‐surface and intracellular proteins, but some proteins such as cytokines may be transient and difficult to measure. Thus, a flow cytometry method that can simultaneously measure cytokine mRNA and protein levels in single cells is a very powerful tool. We defined a flow cytometry method that combines the conventional measurement of T cell surface proteins (CD45, CD3, CD4, CD8) and intracellular cytokines (IL‐2, INF‐γ) with fluorescent in situ hybridization and branched DNA technology for amplification and detection of IL‐2 and INF‐γ mRNA transcripts in activated T cells. This method has been applied to frozen peripheral mononuclear blood cells (PBMCs) and frozen blood samples, making it applicable to clinical trial specimens that require shipment to the test site. In CD4+ cells from activated PBMCs, the concordance between mRNA and protein levels was 41% for IL‐2 and 21% for and INF‐γ. In CD8+ cells from activated PBMCs, the concordance was 15% for IL‐2 and 32% for INF‐γ. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Detection of IL‐2 and IFN‐γ mRNA and protein expression in frozen PBMCs Alternate Protocol: Detection of IL‐2 and IFN‐γ mRNA and protein expression in frozen blood

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Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

Cited by 105 publications

References 28 publications

Reference gene selection for quantitative PCR studies in bovine neutrophils

Reference gene selection for quantitative PCR studies in bovine neutrophils

Quantification of HIV-1 latency reversal in resting CD4 ⁺ T cells from patients on suppressive antiretroviral therapy

Single‐Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry

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Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

Cited by 105 publications

References 28 publications

Reference gene selection for quantitative PCR studies in bovine neutrophils

Reference gene selection for quantitative PCR studies in bovine neutrophils

Quantification of HIV-1 latency reversal in resting CD4 + T cells from patients on suppressive antiretroviral therapy

Single‐Cell Analysis of Cytokine mRNA and Protein Expression by Flow Cytometry

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Product

Resources

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Quantification of HIV-1 latency reversal in resting CD4 ⁺ T cells from patients on suppressive antiretroviral therapy