Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays

Luzzi, Veronica; Holtschlag, Victoria; Watson, Mark A.

doi:10.1038/87354

Cited by 33 publications

(50 citation statements)

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“…T7-based linear amplification was used to this end and was shown to preserve the expression profile. 9,10,24 However, this technique is labor-intensive, long-lasting, and because of multiple steps highly susceptible to the slightest influences. Calculating an amplification factor of ϳ10 to 50 per working cycle, it has to be repeated two or three times, including several incubation and cleaning steps, thereby exposing the RNA to the danger of loss and degradation.…”

Section: Discussionmentioning

confidence: 99%

cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue

Fink

Kohlhoff

Stein

et al. 2002

The American Journal of Pathology

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Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to ϳ10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs (ϳ500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from

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Section: Discussionmentioning

confidence: 99%

cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue

Fink

Kohlhoff

Stein

et al. 2002

The American Journal of Pathology

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“…Biotinylated cRNA target was generated as previously described 15 from both amplified and nonamplified cDNAs using the Bioarray high-yield transcription kit (ENZO, New York, NY) following the manufacturer's protocol. After a 5-hr incubation period at 37°C, the final biotin-labeled cRNA product was purified using RNeasy spin columns (Qiagen) and eluted twice in 30 ll of RNase-free water.…”

Section: Biotin-labeled Crna Transcriptionmentioning

confidence: 99%

Global analysis of host tissue gene expression in the invasive front of colorectal liver metastases

Bandapalli

Geheeb

Kobelt

et al. 2005

Intl Journal of Cancer

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Host cell reactions are a crucial determinant for tumor invasion. We analyzed on a genomewide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross‐hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. Using the gene ontology (GO) classification, we were able to determine patterns of up‐ and downregulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation, e.g., of the GO terms “extracellular matrix,” “cell communication,” “response to biotic stimulus,” “structural molecule activity” and “cell growth,” indicating a very pronounced host cell response to tumor invasion. On the single gene level, hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the noninvaded liver tissue. In summary, our data demonstrate the feasibility of an interspecies differential gene expression approach on a genomewide scale. © 2005 Wiley‐Liss, Inc.

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“…This approach, with amplification efficiencies of more than 1000-fold, has been used for laser microdissected tissue from frozen sections (64,65). A major concern is the linearity of the amplification across different mRNA species.…”

Section: Sensitive Assays For Renal Gene Expression Have Been Developedmentioning

confidence: 99%

Repuncturing the Renal Biopsy

Kretzler¹,

Cohen²,

Doran³

et al. 2002

Journal of the American Society of Nephrology

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Expression profiling of ductal carcinoma in situ by laser capture microdissection and high-density oligonucleotide arrays

Cited by 33 publications

References 0 publications

cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue

cDNA Array Hybridization after Laser-Assisted Microdissection from Nonneoplastic Tissue

Global analysis of host tissue gene expression in the invasive front of colorectal liver metastases

Repuncturing the Renal Biopsy

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