Selection and validation of endogenous reference genes using a high throughput approach

Jin, Ping; Zhao, Yingdong; Ngalame, Yvonne; Panelli, Monica C.; Nagorsen, Dirk; Monsurró, Vladia; Smith, Kina; Hu, Nan; Su, Hua; Taylor, Phil R.; Marincola, Francesco M.; Wang, Ena

doi:10.1186/1471-2164-5-55

Cited by 57 publications

(43 citation statements)

References 51 publications

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“…To avoid a potential bias of quantification against a single housekeeping gene, we selected four different transcripts (HPRT1, CBFB, GAPDH and RPL32) for normalisation purposes (32,33). This group of control transcripts was selected because (1) they showed <10% variability in all of our microarray experiments (data not shown); (2) they were expressed at different steady-state levels and (3) they belong to different metabolic pathways and are thus unlikely to be co-regulated.…”

Section: Resultsmentioning

confidence: 99%

The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the Nonsense Mediated Decay pathway

Viegas

Gehring

Breit

et al. 2007

Nucleic Acids Research

111

115

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Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. NMD has been shown to also regulate normal gene expression and thus emerged as one of the key post-transcriptional mechanisms of gene regulation. Recently, NMD efficiency has been shown to vary between cell types and individuals thus implicating NMD as a modulator of genetic disease severity. We have now specifically analysed the molecular mechanism of variable NMD efficiency and first established an assay system for the quantification of NMD efficiency, which is based on carefully validated cellular NMD target transcripts. In a HeLa cell model system, NMD efficiency is shown to be remarkably variable and to represent a stable characteristic of different strains. In one of these strains, low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in this model. We conclude that cellular concentrations of RNPS1 can modify NMD efficiency and propose that cell type specific co-factor availability represents a novel principle that controls NMD.

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Section: Resultsmentioning

confidence: 99%

The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the Nonsense Mediated Decay pathway

Viegas

Gehring

Breit

et al. 2007

Nucleic Acids Research

111

115

View full text Add to dashboard Cite

show abstract

“…Microarray data were uploaded to the mAdb databank (http://nciarray.nci.nih.gov) and further analysed using Partek (St. Louis, Missouri, USA) and BRBArrayTools developed by the Biometric Research Branch, NCI (http://linus.nci.nih.gov/BRB-ArrayTools.html) [25]. The whole gene expression profile consisted of 25100 genes.…”

Section: Methodsmentioning

confidence: 99%

“…Survival analysis, survival risk prediction analysis and Support Vector Machine (SVM) were performed using BRBArrayTools [25]. Univariant Cox proportional hazards models were fit to test individual NK selected gene expression levels for association with survival (Wald test, two-sided).…”

Section: Methodsmentioning

confidence: 99%

Molecular signatures mostly associated with NK cells are predictive of relapse free survival in breast cancer patients

et al. 2013

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BackgroundRecent observations suggest that immune-mediated tissue destruction is dependent upon coordinate activation of immune genes expressed by cells of the innate and adaptive immune systems.MethodsHere, we performed a retrospective pilot study to investigate whether the coordinate expression of molecular signature mostly associated with NK cells could be used to segregate breast cancer patients into relapse and relapse-free outcomes.ResultsBy analyzing primary breast cancer specimens derived from patients who experienced either 58–116 months (~5-9 years) relapse-free survival or developed tumor relapse within 9–76 months (~1-6 years) we found that the expression of molecules involved in activating signaling of NK cells and in NK cells: target interaction is increased in patients with favorable prognosis.ConclusionsThe parameters identified in this study, together with the prognostic signature previously reported by our group, highlight the cooperation between the innate and adaptive immune components within the tumor microenvironment.

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“…Both, human reference and test total RNA were amplified into antisense RNA in large amounts using identical conditions [14, 15]. Confidence about array quality was confirmed as previously described [16]. For 36 k human array performances, both reference and test aRNA were directly labeled using ULS aRNA Fluorescent Labeling kit (Kreatech) with Cy3 for reference and Cy5 for test samples.…”

Section: Methodsmentioning

confidence: 99%

A signature of immune function genes associated with recurrence-free survival in breast cancer patients

Ascierto

Kmieciak

Idowu

et al. 2011

Breast Cancer Res Treat

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153

150

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The clinical significance of tumor-infiltrating immune cells has been reported in a variety of human carcinomas including breast cancer. However, molecular signature of tumor-infiltrating immune cells and their prognostic value in breast cancer patients remain elusive. We hypothesized that a distinct network of immune function genes at the tumor site can predict a low risk versus high risk of distant relapse in breast cancer patients regardless of the status of ER, PR, or HER-2/neu in their tumors. We conducted retrospective studies in a diverse cohort of breast cancer patients with a 1–5 year tumor relapse versus those with up to 7 years relapse-free survival. The RNAs were extracted from the frozen tumor specimens at the time of diagnosis and subjected to microarray analysis and real-time RT-PCR. Paraffin-embedded tissues were also subjected to immunohistochemistry staining. We determined that a network of immune function genes involved in B cell development, interferon signaling associated with allograft rejection and autoimmune reaction, antigen presentation pathway, and cross talk between adaptive and innate immune responses were exclusively upregulated in patients with relapse-free survival. Among the 299 genes, five genes which included B cell response genes were found to predict with >85% accuracy relapse-free survival. Real-time RT-PCR confirmed the 5-gene prognostic signature that was distinct from an FDA-cleared 70-gene signature of MammaPrint panel and from the Oncotype DX recurrence score assay panel. These data suggest that neoadjuvant immunotherapy in patients with high risk of relapse may reduce tumor recurrence by inducing the immune function genes.

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Selection and validation of endogenous reference genes using a high throughput approach

Cited by 57 publications

References 51 publications

The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the Nonsense Mediated Decay pathway

The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the Nonsense Mediated Decay pathway

Molecular signatures mostly associated with NK cells are predictive of relapse free survival in breast cancer patients

A signature of immune function genes associated with recurrence-free survival in breast cancer patients

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