Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer

Kube, Dagmar M.; Savcı-Heijink, C. Dilara; Lamblin, Anne Françoise; Kosari, Farhad; Vasmatzis, George; Cheville, John C.; Connelly, Donald P.; Klee, George G.

doi:10.1186/1471-2199-8-25

Cited by 63 publications

(58 citation statements)

References 33 publications

(18 reference statements)

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“…This could be due to active RNases present in the tissue and/or improper dehydration leading to RNA degradation. Together with recent results using similar methodology and FR specimens [12,25], our data demonstrate that a modified version of the Arcturus staining method combined with the Arcturus extraction method is highly suitable for morphological assessment and high-quality RNA extraction from LCM-obtained cervical keratinocytes (Fig. 2).…”

Section: Discussionsupporting

confidence: 83%

“…Although the amount of tissue used was in the range specified by the Ambion and Sigma kits, highquality RNA was only obtained using the Arcturus kit, which is designed for extraction from microdissected samples. This indicates that for extraction from small amounts of cervical tissue, as seen previously in prostate tissue [25], methods optimized for extraction from microdissected samples produced favorable results.…”

Section: Discussionsupporting

confidence: 61%

“…Recent evidence suggested that inclusion of an RNase inhibitor [25], as well as a prolonged 100% (v/v) ethanol incubation [11,25], generated higher-quality RNA from microdissected samples. It was previously argued that the protocol described in the Arcturus Histogene Frozen Section Staining Kit is not adequate for obtaining high-quality RNA from FR laser capture microdissected samples [11].…”

Section: Discussionmentioning

confidence: 99%

“…79254) and slight modifications when using tissue sections [25]. Briefly, 100 μl of extraction buffer (XB) was added to samples and incubated at 42 °C for 30 min.…”

Section: Rna Extractionmentioning

confidence: 99%

“…Briefly, 1× ProtectRNA (ProtectRNA RNase Inhibitor 500× concentrate, Catalog No. R7397, SigmaAldrich) was added to the staining solution [25], and tissue was dehydrated as normal with the addition of an extra 100% (v/v) ethanol step to acquire intact RNA [11]. Tissue sections were allowed to dry for up to 3 h prior to capturing 5000-25,000 cells in 1000-5000 captures using CapSure Macro LCM caps.…”

Section: Rna Integrity Assessmentmentioning

confidence: 99%

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Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

DeCarlo

Escott

Werner

et al. 2008

Analytical Biochemistry

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Optimal sample handling techniques for tissue preparation and storage, RNA extraction and quantification, and target gene detection are crucial for reliable gene expression analysis. Methods for measuring low-expressing genes, such as interferons, in human cervical samples are not described in the scientific literature. To detect interferon mRNA in human cervical samples we obtained normal and dysplastic frozen and formalin-fixed cervical biopsies from colposcopy. Histopathological diagnosis was performed by one pathologist. Cervical keratinocytes were isolated using laser capture microdissection. Immortalized keratinocytes transduced with or devoid of an HPV oncogene were used for initial method development. RNA from samples was extracted and integrity tested to compare tissue storage and extraction methods. The expression of five housekeeping genes was analyzed in cell lines and patient tissue to permit normalization between samples using quantitative real-time polymerase chain reaction. The usefulness of cDNA amplification was assessed for the detection of low-expressing interferon κ in cervical tissue. Here we report optimal tissue storage conditions, RNA extraction, sample normalization, and transcript amplification, as well as the sensitivity of quantitative real-time polymerase chain reaction and laser capture microdissection, for interferon κ detection in cervical tissue. Without these optimized techniques, interferon κ detection would be unattainable in cervical samples. KeywordsCervical keratinocytes; Frozen and formalin-fixed cervical tissue; Housekeeping genes; Interferons; Interferon κ; Laser capture microdissection; Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) 1 for the molecular analysis of disease is a powerful and widely used tool. Extraction of high-quality RNA for use in gene expression analysis techniques such as reverse transcription (RT), qRT-PCR, and cDNA microarrays is of great importance. Although obtaining high-quality RNA from cell lines is relatively straightforward, the complexity and heterogeneity of human tissue pose considerable challenges for linking gene expression patterns with disease state. Nonetheless,

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Section: Discussionsupporting

confidence: 83%

Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 99%

“…79254) and slight modifications when using tissue sections [25]. Briefly, 100 μl of extraction buffer (XB) was added to samples and incubated at 42 °C for 30 min.…”

Section: Rna Extractionmentioning

confidence: 99%

Section: Rna Integrity Assessmentmentioning

confidence: 99%

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Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

DeCarlo

Escott

Werner

et al. 2008

Analytical Biochemistry

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CD200 Upregulation in Vascular Endothelium Surrounding Cutaneous Squamous Cell Carcinoma

Belkin¹,

Mitsui²,

Wang³

et al. 2013

JAMA Dermatol

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Gene expression analyses of neurons, astrocytes, and oligodendrocytes isolated by laser capture microdissection from human brain: Detrimental effects of laboratory humidity

Ordway¹,

Szebeni²,

Duffourc³

et al. 2009

J of Neuroscience Research

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Laser capture microdissection (LCM) is a versatile computer-assisted dissection method that permits collection of tissue samples with a remarkable level of anatomical resolution. LCM’s application to the study of human brain pathology is growing, although it is still relatively underutilized, compared with other areas of research. The present study examined factors that affect the utility of LCM, as performed with an Arcturus Veritas, in the study of gene expression in the human brain using frozen tissue sections. LCM performance was ascertained by determining cell capture efficiency and the quality of RNA extracted from human brain tissue under varying conditions. Among these, the relative humidity of the laboratory where tissue sections are stained, handled, and submitted to LCM had a profound effect on the performance of the instrument and on the quality of RNA extracted from tissue sections. Low relative humidity in the laboratory, i.e., 6–23%, was conducive to little or no degradation of RNA extracted from tissue following staining and fixation and to high capture efficiency by the LCM instrument. LCM settings were optimized as described herein to permit the selective capture of astrocytes, oligodendrocytes, and noradrenergic neurons from tissue sections containing the human locus coeruleus, as determined by the gene expression of cell-specific markers. With due regard for specific limitations, LCM can be used to evaluate the molecular pathology of individual cell types in post-mortem human brain.

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Optimization of laser capture microdissection and RNA amplification for gene expression profiling of prostate cancer

Cited by 63 publications

References 33 publications

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

Gene expression analysis of interferon κ in laser capture microdissected cervical epithelium

CD200 Upregulation in Vascular Endothelium Surrounding Cutaneous Squamous Cell Carcinoma

Gene expression analyses of neurons, astrocytes, and oligodendrocytes isolated by laser capture microdissection from human brain: Detrimental effects of laboratory humidity

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