Both β‐actin and GAPDH are useful reference genes for normalization of quantitative RT‐PCR in human FFPE tissue samples of prostate cancer

Mori, Rintaro; Wang, Qingcai; Danenberg, Kathleen D.; Pinski, Jacek; Danenberg, Peter V.

doi:10.1002/pros.20815

Cited by 104 publications

(80 citation statements)

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“…Overall, our study demonstrates that UBC, YW-HAZ, RPLP and TBP, were the most suitable reference genes to be used for gene expression studies using MCF7, HCT116 and HepG2 cells. Although GAPDH and ACTB are used as reference genes in most in vitro studies using these cell lines (Bracke et al 1993;Tanic et al 2007;Cicinnati et al 2008;Mori et al 2008), these two reference genes did not exhibit stable expression levels across MCF7, HCT116 and HepG2 cells, as demonstrated by our current results as well as previous studies (Schmittgen and Zakrajsek 2000;Gorzelniak et al 2001). The GAPDH and ACTB genes are variably expressed when subjected to serumstimulation (Schmittgen and Zakrajsek 2000) and hormone treatments (Gorzelniak et al 2001).…”

Section: Discussionsupporting

confidence: 71%

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

et al. 2011

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Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of C t values. The geNorm analysis showed the following ranking for stability of genes:A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines.

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Section: Discussionsupporting

confidence: 71%

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

et al. 2011

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“…Aunque en el estudio de Tomlins et al, (2005), se utilizó el gen GAPDH para determinar de manera indirecta la cantidad e integridad del RNA y en este estudio además del GAPDH se utilizó el UBC, ambos asociados a estudios de CaP (Mori et al, 2008;Rose et al, 2005); el tamaño del amplímero de estos dos genes (87 y 133 pb, respectivamente) es inferior al esperado en la fusión 2, lo cual hace necesaria la confirmación de los casos negativos empleando un gen de referencia de mayor tamaño que demostrara que el RNA no está degradado.…”

Section: Discussionunclassified

Estandarización del protocolo para la detección de las fusiones TMPRSS2:ERG y de la expresión de los genes EZH2, SPINK-1 y NKX3.1 en cáncer de próstata (CaP)

Moreno

López

2016

Acta biol. Colomb.

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Associate Editor: Argel Aguilar-Valles.Citation/Citar este artículo como: Segura Moreno YY, Serrano López ML. Estandarización del protocolo para la detección de las fusiones TMPRSS2:ERG y de la expresión de los genes EZH2, SPINK-1 y NKX3.1 en cáncer de próstata (CaP RESUMENEn la actualidad no existe una herramienta que permita diferenciar pacientes con cáncer de próstata (CaP) de mal pronóstico de aquellos con enfermedad indolente que sólo requieren un seguimiento controlado de la enfermedad. Debido a la coexistencia de diferentes focos premalignos y malignos en el CaP, el entendimiento sobre el proceso de carcinogénesis requiere de un mejor conocimiento. Actualmente, la heterogeneidad morfológica en CaP es evaluada con la puntuación de Gleason, la cual está fuertemente relacionada con el pronóstico de la enfermedad, sin embargo, esto es insuficiente por lo que se trabaja actualmente en identificación de alteraciones moleculares que permitan identificar subtipos que puedan establecer de manera más precisa el pronóstico del paciente. Este estudio preliminar buscó la estandarización del método de cuantificación en muestras prostáticas de FFPE de la expresión de los transcritos de posibles biomarcadores, como los oncogenes SPINK-1 y EZH2, el supresor tumoral NKX3.1, en conjunto con la determinación de la presencia/ausencia del gen de fusión TMPRSS2:ERG, ya que estos transcritos se encuentran involucrados en aparentes eventos excluyentes de la evolución natural del CaP, que apoyan la posibilidad de una clasificación molecular para esta enfermedad.Palabras clave: expresión génica, neoplasia intraepitelial prostática, neoplasias de próstata, progresión de la enfermedad, puntuación de Gleason. ABSTRACTAt present doesn't exist tool to differentiate patients with prostate cancer (PCa) of poor prognosis of those with indolent disease that only require a controlled monitoring of the disease. Because of the coexistence of different premalignant and malignant foci in CaP, the understanding of the carcinogenesis process requires a better understanding. Currently, the morphological heterogeneity in PCa is evaluated with Gleason score, which is closely related to the prognosis of the disease, but this is insufficient so it is currently to work on identifying molecular alterations to identify subtypes that can establish more precisely the patient's prognosis. This preliminary study aimed to standardization of the method of quantification in prostatic samples of FFPE of expression of transcripts of possible biomarkers, such as the oncogenes, SPINK-1 y EZH2, the tumour suppressor, NKX3.1, together with the determination of the presence/absence of gene fusion, TMPRSS2:ERG, being that these transcripts are involved in apparent exclusive events of the natural evolution of PCa, that support the possibility of a molecular classification for this disease.

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“…Uma análise de bootstrapping com 1000 interações foi realizada utilizando-se SEQBOOT (Felsenstein, 1993 (Mori et al, 2008). As condições da PCR também seguiram os procedimentos já descritos, e usaram-se os primers sbGnRH 05 e 06 e BAC 01 e 02 (Tab.…”

Section: *Primers Degenerados: M (A + C) W (A + T) R (A + G) K (T unclassified

Clonagem e avaliação da expressão gênica do sbGnRH em machos juvenis e adultos de linguado, Paralichthys orbignyanus

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et al. 2011

Arq. Bras. Med. Vet. Zootec.

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Both β‐actin and GAPDH are useful reference genes for normalization of quantitative RT‐PCR in human FFPE tissue samples of prostate cancer

Abstract: The good correlation between gene expression values obtained when using beta-actin and GAPDH as reference genes suggests that either gene is a valid denominator for gene expression studies in prostate cancer.

Cited by 104 publications

References 32 publications

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

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

Estandarización del protocolo para la detección de las fusiones TMPRSS2:ERG y de la expresión de los genes EZH2, SPINK-1 y NKX3.1 en cáncer de próstata (CaP)

Clonagem e avaliação da expressão gênica do sbGnRH em machos juvenis e adultos de linguado, Paralichthys orbignyanus

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