Generation of a non-small cell lung cancer transcriptome microarray

Tanney, Austin; Oliver, Gavin R.; Farztdinov, V. M.; Kennedy, Richard D.; Mulligan, Jude M.; Fulton, C. E.; Farragher, Susan; Field, John K.; Johnston, Patrick G.; Harkin, D. Paul; Proutski, Vitali; Mulligan, Karl

doi:10.1186/1755-8794-1-20

Cited by 19 publications

(25 citation statements)

References 39 publications

(34 reference statements)

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“…This possibility cannot be excluded on the basis of the experimental design and the controls included here. Because the number of cells recovered by flow cytometry was relatively small (ϳ5 ϫ 10 5 ), we used linear enzymatic amplification (6,25,30,33) to generate sufficient RNA for microarray hybridization. Before and after RNA amplification, portions of the same RNA samples as those analyzed with microarrays were used as the template for RT-PCR.…”

Section: Discussionmentioning

confidence: 99%

“…HCT-8 cells seeded in T25 flasks, or 96-well plates were infected with C. parvum oocysts at a 1:1, 1:2, 1:4, or 1:50 oocyst/host cell ratio and incubated for 6,19,24,30,43,48,72, and 88 h as described above. Monolayers were released as described above, washed three times with PBS, and incubated with fluorescein-labeled lectins (Vector Laboratories, Burlingame, CA) at 37°C for 30 min (10) before or after the permeabilization of cell membranes with 100% methanol for 15 min.…”

Section: Methodsmentioning

confidence: 99%

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Cell Sorting-Assisted Microarray Profiling of Host Cell Response to Cryptosporidium parvum Infection

2010

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To study the transcriptional response of mammalian cells to infection with the intracellular apicomplexan parasite Cryptosporidium parvum, infected and uninfected cells were recovered from C. parvum-infected cell monolayers. This approach, which contrasts with a more conventional experimental design that compares infected to uninfected cell monolayers, enabled the identification of functional categories of genes that are differentially transcribed as a direct consequence of the presence of intracellular parasites. Among several categories of upregulated genes, glycoprotein metabolism was significantly overrepresented. To investigate whether these transcriptional changes affected the composition of the surface of infected cells, cells were probed with fluorescently labeled lectins. Among a panel of seven lectins, soybean agglutinin, which recognizes N-acetyl-D-galactosamine, generated the largest difference in fluorescence between infected and uninfected cells. The origin of the fluorescent signal emitted by infected cells was further investigated and attributed to the overexpression of glycoprotein on the surface of infected cells, as well as the presence of glycoprotein located in the proximity of intracellular parasites.Cryptosporidium parvum, an apicomplexan protozoan species, infects the intestine and sometimes the respiratory tract and bile duct of various mammalian hosts. In immunocompetent humans the infection causes self-limited diarrhea, but in immunocompromised individuals, primarily those with AIDS, the infection can become chronic and potentially cause lifethreatening syndromes (5). Infection with Cryptosporidium parasites occurs when food or water contaminated with oocysts is ingested, or possibly through the inhalation of oocysts. After entering the gastrointestinal tract of the host, four sporozoites released from the oocyst seek to invade the intestinal epithelial cells, where they multiply as meronts.Global gene expression profiles in cell monolayers infected with C. parvum have been studied using microarrays (3,8,20). These studies found that genes belonging to the cell proliferation, apoptosis, signal transduction, and transcription categories are overrepresented among significantly regulated genes (8,20,39). The overexpression of the osteoprotegerin gene in host cells after infection also was reported (3).Published microarray studies of C. parvum-infected cells are based on RNA extracted from cells in culture. The development of this parasite in culture is restricted in time, and the completion of the life cycle is only rarely observed (14, 32). High oocyst doses and long incubation times do not increase the proportion of infected cells (36) and may instead lead to the extensive perturbation of the monolayer caused primarily by apoptosis and mitosis (19,21,34,38). Discerning which transcriptional changes occur directly in response to the infection, and which result from the perturbance of the monolayer (13, 36), is difficult. To eliminate the effect of partial infection and monolayer perturba...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cell Sorting-Assisted Microarray Profiling of Host Cell Response to Cryptosporidium parvum Infection

2010

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“…Total RNA was amplified using the NuGEN WT-Ovation FFPE System (NuGEN, San Carlos, CA) and hybridized to the Almac Breast Cancer DSA (Affymetrix, Santa Clara, CA) as described previously (19,20). …”

Section: Methodsmentioning

confidence: 99%

Identification and Validation of an Anthracycline/Cyclophosphamide–Based Chemotherapy Response Assay in Breast Cancer

Mulligan¹,

Hill²,

Deharo³

et al. 2014

JNCI: Journal of the National Cancer Institute

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BackgroundThere is no method routinely used to predict response to anthracycline and cyclophosphamide–based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection.MethodsDNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided.ResultsIn a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population.ConclusionsA formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide–based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.

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“…Almac have developed a range of high density Disease SpeciWc MicroArrays (DSAs™) manufactured on the gold standard AVymetrix platform, which capture as completely as possible all transcripts transcribed in a speciWc disease setting such as breast, colorectal or nonsmall cell lung cancer. The actual content of the DSA is derived using a combination of high throughput 3Ј-based sequencing, data mining and expression analysis (Tanney et al 2008) (Fig. 4).…”

Section: Dna Microarray Analysis Of Ffpe Samplesmentioning

confidence: 99%

RNA expression analysis from formalin fixed paraffin embedded tissues

Farragher

Tanney

Kennedy

et al. 2008

Histochem Cell Biol

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168

147

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Formalin fixation and paraffin embedding (FFPE) is the most commonly used method worldwide for tissue storage. This method preserves the tissue integrity but causes extensive damage to nucleic acids stored within the tissue. As methods for measuring gene expression such as RT-PCR and microarray are adopted into clinical practice there is an increasing necessity to access the wealth of information locked in the Formalin fixation and paraffin embedding archives. This paper reviews the progress in this field and discusses the unique opportunities that exist for the application of these techniques in the development of personalized medicine.

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Generation of a non-small cell lung cancer transcriptome microarray

Cited by 19 publications

References 39 publications

Cell Sorting-Assisted Microarray Profiling of Host Cell Response to Cryptosporidium parvum Infection

Cell Sorting-Assisted Microarray Profiling of Host Cell Response to Cryptosporidium parvum Infection

Identification and Validation of an Anthracycline/Cyclophosphamide–Based Chemotherapy Response Assay in Breast Cancer

RNA expression analysis from formalin fixed paraffin embedded tissues

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