Selection of reference genes for quantitative polymerase chain reaction studies in purified B cells from B cell chronic lymphocytic leukaemia patients

Valceckiene, Vilma; Kontenyte, Rima; Jakubauskas, Artūras; Griškevičius, Laimonas

doi:10.1111/j.1365-2141.2010.08363.x

Cited by 18 publications

(14 citation statements)

References 28 publications

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“…β-2-microglobulin ( B2M ) is a component of major histocompatibility complex I, hence being expressed in every nucleated cell, and has yet been applied before as a normalization scalar in different set-ups [26-28]. Ribosomal protein L13A ( RPL13A ) is involved in the process of transcript translation, and has also been widely included as a reference gene for RT-qPCR analyses [29-32], in spite of possible presence of pseudogenes [33].…”

Section: Discussionmentioning

confidence: 99%

Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells

et al. 2013

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BackgroundSelecting stably expressed reference genes is essential for proper reverse transcription quantitative polymerase chain reaction gene expression analysis. However, this choice is not always straightforward. In the case of differentiating human embryonic stem (hES) cells, differentiation itself introduces changes whereby reference gene stability may be influenced.ResultsIn this study, we evaluated the stability of various references during retinoic acid-induced (2 microM) differentiation of hES cells. Out of 12 candidate references, beta-2-microglobulin, ribosomal protein L13A and Alu repeats are found to be the most stable for this experimental set-up.ConclusionsOur results show that some of the commonly used reference genes are actually not amongst the most stable loci during hES cell differentiation promoted by retinoic acid. Moreover, a novel normalization strategy based on expressed Alu repeats is validated for use in hES cell experiments.

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Section: Discussionmentioning

confidence: 99%

Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells

et al. 2013

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“…A major finding of our study was that several conventional "housekeeping genes" proved to be unreliable controls, which is in line with previous reports about an unstable expression of commonly used reference genes, such as GAPDH , ACTB or HPRT1 , in various experimental setups [11,20-22]. Of note, IPO8 and ACTB behaved considerably differently regarding their stability in neutrophils or T cells, and candidate genes we found inappropriate for normalization in activated T cells have been reported to be stably expressed in LPS-treated monocytes ( B2M , PPIA , ACTB [11]) or B cells from chronic lymphocytic leukemia patients ( B2M , HPRT1 [23]). These findings underscore the necessity of careful individual validation of reference genes for every leukocyte subtype and every experimental condition.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2011

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BackgroundThe choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR). This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes.ResultsThe mRNA expression of 17 (T cells), 7 (neutrophils) or 8 (unselected leukocytes) potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells.ConclusionsThe study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

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“…For realtime RT-PCR three loading controls – HPRT1, GUSB , and B2M were selected based on prior work by Valceckiene et al 20 . The primer probe sets recognizing these three loading controls and Glucose Transporter ( GLUT ) 1, 3, and 4 were purchased from Applied Biosystems.…”

Section: Methodsmentioning

confidence: 99%

Investigating and targeting chronic lymphocytic leukemia metabolism with the human immunodeficiency virus protease inhibitor ritonavir and metformin

Adekola

Dalva-Aydemir

et al. 2014

Leukemia & Lymphoma

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Chronic Lymphocytic Leukemia (CLL) remains fatal due to the development of resistance to existing therapies. Targeting abnormal glucose metabolism sensitizes various cancer cells to chemotherapy and/or elicits toxicity. Examination of glucose dependency in CLL demonstrated variable sensitivity to glucose deprivation. Further evaluation of metabolic dependencies of CLL cells resistant to glucose deprivation revealed increased engagement of fatty acid oxidation upon glucose withdrawal. Investigation of glucose transporter expression in CLL reveals up-regulation of glucose transporter GLUT4. Treatment of CLL cells with HIV protease inhibitor ritonavir, that inhibits GLUT4, elicits toxicity similar to that elicited upon glucose-deprivation. CLL cells resistant to ritonavir are sensitized by co-treatment with metformin, potentially targeting compensatory mitochondrial complex 1 activity. Ritonavir and metformin have been administered in humans for treatment of diabetes in HIV patients, demonstrating the tolerance of this combination in humans. Our studies strongly substantiate further investigation of FDA approved ritonavir and metformin for CLL.

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Selection of reference genes for quantitative polymerase chain reaction studies in purified B cells from B cell chronic lymphocytic leukaemia patients

Cited by 18 publications

References 28 publications

Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells

Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells

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

Investigating and targeting chronic lymphocytic leukemia metabolism with the human immunodeficiency virus protease inhibitor ritonavir and metformin

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