Genomic DNA functions as a universal external standard in quantitative real-time PCR

Yun, James J.; Heisler, Lawrence E.; Hwang, Irene; Wilkins, Olivia; Lau, Susan; Hyrcza, Martin; Jayabalasingham, Bamini; Jin, Jing; McLaurin, J.; Tsao, Ming‐Sound; Der, Sandy D.

doi:10.1093/nar/gkl400

Cited by 150 publications

(121 citation statements)

References 24 publications

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“…cDNA was created by adding 10 ng RNA to the RT master mix and was reverse transcribed using the Superscript III Kit (Invitrogen). Genomic DNA standards were isolated from purified placental tissue and used as a universal standard over a 7-log dilution range to calculate relative gene-expression levels (25). Single intraexon gene-specific primers were generated using Primer Express Software (Perkin Elmer Applied Biosystems) or OligoPerfect (Invitrogen).…”

Section: Intracellular Cytokine Stainingmentioning

confidence: 99%

Characterization of a Human Cervical CD4+ T Cell Subset Coexpressing Multiple Markers of HIV Susceptibility

McKinnon

Nyanga

Chege

et al. 2011

The Journal of Immunology

161

158

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The HIV pandemic disproportionately affects women, with most infections acquired through receptive vaginal sex. Although the target cells by which HIV establishes infection in the female genital tract remain poorly defined, it is known that immune activation results in CD4+ T cells with enhanced susceptibility, as does expression of the mucosal integrin α4β7 and the HIV coreceptor CCR5. Blood and cervical cytobrush specimens were collected from female sex workers (FSWs) in Nairobi, Kenya. Genital infection diagnostics were performed, T cell populations were defined by multiparameter flow cytometry based on their expression of surface receptors relevant to mucosal homing and/or HIV acquisition, and cytokine production was assayed by intracellular cytokine staining. The integrin α4β7 was expressed on 26.0% of cervical CD4+ T cells, and these cells were more likely to express both the HIV coreceptor CCR5 (p < 0.0001) and the early activation marker CD69 (p < 0.0001) but not CXCR4 (p = 0.34). Cervical Th17 frequencies were enhanced compared with blood (7.02 versus 1.24%; p < 0.0001), and cervical IL-17A+ CD4+ T cells preferentially coexpressed α4β7 and CCR5. Expression of IFN-γ and IL-22 was greater in cervical Th17 cells than in blood Th17 cells. In keeping with the hypothesis that these cells are preferential HIV targets, gp120 preferentially bound CCR5+ cervical T cells, and cervical Th17 cells were almost completely depleted in HIV+ FSWs compared with HIV− FSWs. In summary, a subset of Th17 CD4+ T cells in the cervical mucosa coexpresses multiple HIV susceptibility markers; their dramatic depletion after HIV infection suggests that these may serve as key target cells during HIV transmission.

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Section: Intracellular Cytokine Stainingmentioning

confidence: 99%

Characterization of a Human Cervical CD4+ T Cell Subset Coexpressing Multiple Markers of HIV Susceptibility

McKinnon

Nyanga

Chege

et al. 2011

The Journal of Immunology

161

158

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“…The reactions were activated at 951C for 3 min, followed by 40 cycles of denaturation at 951C for 15 s, annealing at 651C for 15 s and extension at 721C for 20 s. Transcript amount per nanogram of cDNA was obtained from standard curves generated with a pool of 10 non-tumor lung genomic DNAs. 24 Technical replicates were collapsed by averaging. Normal- Figure 1 Immunohistochemistry results for glypican-3.…”

Section: Glypican-3 Mrna Expression Study By Quantitative Polymerase mentioning

confidence: 99%

Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma

et al. 2008

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Glypican-3 is a membrane-bound proteoglycan whose expression has been linked to malignancies through the existence of both mutations and aberrant protein expression. Reports on glypican-3 expression in lung cancer were limited, with some evidence for loss of expression, which suggested a tumor-suppressor role. We sought to evaluate glypican-3 expression in lung cancer at the protein and mRNA levels and correlate it with clinical, histological and genomic characteristics such as RAS mutation status. We used immunohistochemistry on tissue microarray to study glypican-3 expression in 97 patients, evaluated glypican-3 mRNA levels by quantitative polymerase chain reaction in 143 patients and identified RAS mutations by allele-specific oligonucleotide hybridization. We correlated the results with clinical and histological data. Glypican-3 immunostaining was negative in all normal lung tissues, but positive in 23% of lung carcinoma samples. High protein and mRNA expression was associated with squamous histology (positive stain in 55% of squamous cell carcinoma vs 8% of adenocarcinoma, Po0.0001 for both immunostaining and mRNA). RAS mutations were highly associated with adenocarcinoma and low glypican-3 mRNA expression (Po0.0001 for both). Among smokers, glypican-3 mRNA expression was reduced in adenocarcinoma patients (P ¼ 0.013), and was elevated in those with squamous cell carcinoma (P ¼ 0.03, interaction P ¼ 0.0009). These opposing associations also correlated with the smoking burden. Patients with tumors staining positively for glypican-3 smoked significantly more than patients with tumors staining negatively (P ¼ 0.013). No association was found between glypican-3 expression and patient outcome. In conclusion, glypican-3 was overexpressed in cancerous compared with normal lung tissue. Adenocarcinoma and squamous cell carcinoma had differential expression of glypican-3, with predilection to squamous cell carcinoma patients who smoked. Glypican-3 expression in squamous cell carcinoma as an oncofetal protein renders it a potential candidate marker for early detection of lung squamous cell carcinoma.

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“…The reactions were activated at 951C for 3 min followed by 40 cycles of 951C (15 s), 651C (15 s) and 721C (20 s). The amount of transcript per ng cDNA was calculated using standard curves generated using a pool of genomic DNA from 10 normal lung tissue samples as described (Yun et al, 2006). Primer sequences were designed with Primer Express v 2.0 (Applied Biosystems).…”

Section: Quantification and Analysis Of Ddr Expression In Lung Tumourmentioning

confidence: 99%

Expression and mutation analysis of the discoidin domain receptors 1 and 2 in non-small cell lung carcinoma

et al. 2007

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The discoidin domain receptors, (DDR)1 and DDR2, have been linked to numerous human cancers. We sought to determine expression levels of DDRs in human lung cancer, investigate prognostic determinates, and determine the prevalence of recently reported mutations in these receptor tyrosine kinases. Tumour samples from 146 non-small cell lung carcinoma (NSCLC) patients were analysed for relative expression of DDR1 and DDR2 using quantitative real-time PCR (qRT-PCR). An additional 23 matched tumour and normal tissues were tested for differential expression of DDR1 and DDR2, and previously reported somatic mutations. Discoidin domain receptor 1 was found to be significantly upregulated by 2.15-fold (P ¼ 0.0005) and DDR2 significantly downregulated to an equivalent extent (P ¼ 0.0001) in tumour vs normal lung tissue. Discoidin domain receptor 2 expression was not predictive for patient survival; however, DDR1 expression was significantly associated with overall (hazard ratio (HR) 0.43, 95% CI ¼ 0.22 -0.83, P ¼ 0.014) and disease-free survival (HR ¼ 0.56, 95% CI ¼ 0.33 -0.94, P ¼ 0.029). Multivariate analysis revealed DDR1 is an independent favourable predictor for prognosis independent of tumour differentiation, stage, histology, and patient age. However, contrary to previous work, we did not observe DDR mutations. We conclude that whereas altered expression of DDRs may contribute to malignant progression of NSCLC, it is unlikely that this results from mutations in the DDR1 and DDR2 genes that we investigated.

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Genomic DNA functions as a universal external standard in quantitative real-time PCR

Cited by 150 publications

References 24 publications

Characterization of a Human Cervical CD4+ T Cell Subset Coexpressing Multiple Markers of HIV Susceptibility

Characterization of a Human Cervical CD4+ T Cell Subset Coexpressing Multiple Markers of HIV Susceptibility

Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma

Expression and mutation analysis of the discoidin domain receptors 1 and 2 in non-small cell lung carcinoma

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