Evaluation of Global RNA Amplification and Its Use for High-Throughput Transcript Analysis of Laser-Microdissected Endosperm

Day, Robert C.; McNoe, Les; Macknight, Richard C.

doi:10.1155/2007/61028

Cited by 23 publications

(30 citation statements)

References 74 publications

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“…end. This template is then used by T7 RNA polymerase to generate copies of the cDNA template (Day et al 2007). PCR-based procedures for RNA amplification have also been used, enabling much greater yields per round of amplification (Iscove et al 2002).…”

Section: Application Of Lm In Plant Biology: Gene Expression Studies mentioning

confidence: 99%

“…The PCR-based method in fact produces exponentially amplified cDNA template that then can be used for downstream analyses and, for this reason, less starting material can be used to generate enough target for high throughput analyses using only one round of amplification. PCR-and IVT-based amplification methods have recently been evaluated by Day et al (2007) in microarray experiments on laser microdissected endosperm. Starting from only 50 ng of RNA, amplification methods produced sufficient product for microarray hybridizations, with two-round IVT giving the best results and allowing the identification of endosperm enriched marker genes (Day et al 2007).…”

Section: Application Of Lm In Plant Biology: Gene Expression Studies mentioning

confidence: 99%

“…PCR-and IVT-based amplification methods have recently been evaluated by Day et al (2007) in microarray experiments on laser microdissected endosperm. Starting from only 50 ng of RNA, amplification methods produced sufficient product for microarray hybridizations, with two-round IVT giving the best results and allowing the identification of endosperm enriched marker genes (Day et al 2007). A T7 RNA polymerase-based RNA amplification was recently used to generate sufficient cDNA for 454 sequencing from RNA of maize SAM (Shoot Apical Meristem) cells isolated by LM (Emrich et al 2007).…”

Section: Application Of Lm In Plant Biology: Gene Expression Studies mentioning

confidence: 99%

See 2 more Smart Citations

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

Balestrini¹,

Gómez-Ariza²,

Klink

et al. 2009

Journal of Plant Interactions

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Laser Microdissection (LM) is a technology that allows the rapid procurement of selected cell populations from a section of heterogeneous tissues in a manner conducive to the extraction of DNA, RNA, proteins and even metabolites. In the past few years, it has also been applied to plant biology in order to study gene expression in plant-nematode and plant-microbe interactions. LM represents a powerful tool since cells associated with a particular infection stage can be visualized under the microscope and harvested. Therefore, verification of the response of the plant during the progression of the colonization can be performed in different cell types. Applications of LM to study the interaction between the plant and both pathogenic and symbiotic organisms (i.e. nematode and fungi, respectively) are explored in this review.

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Section: Application Of Lm In Plant Biology: Gene Expression Studies mentioning

confidence: 99%

Section: Application Of Lm In Plant Biology: Gene Expression Studies mentioning

confidence: 99%

Section: Application Of Lm In Plant Biology: Gene Expression Studies mentioning

confidence: 99%

See 1 more Smart Citation

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

Balestrini¹,

Gómez-Ariza²,

Klink

et al. 2009

Journal of Plant Interactions

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“…Although normalization of qRT-PCR-derived expression levels requires an appropriate reference or housekeeping gene (HKG) for compensating for differences between samples [15,16], HKG validation is required for each tissue type [17]. Furthermore, low transcript amounts represent an obstacle for simultaneous analysis of multiple targets or lowexpressing genes, and thus, several methods exist for amplifying mRNA transcripts extracted from tissue samples [18][19][20][21]. However, none of these parameters have been described or optimized for gene expression analysis in cervical samples.…”

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confidence: 99%

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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“…5 Ten kinesins with endosperm preferred expression fell (based on the Operon V1.0 Arabidopsis set). 3 The procedure identified 2,568 individual loci as being preferentially expressed in the endosperm and further analysis showed approximately 800 of these to be early seed-specific. This data has been further validated by quantitative reverse transcription PCR and by using GUS reporter lines.…”

Section: Phragmoplast Formationmentioning

confidence: 99%

Identification of cytoskeleton-associated genes expressed during Arabidopsis syncytial endosperm development

Day

Müller

Macknight

2009

Plant Signaling & Behavior

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During the early stages of Arabidopsis seed development, the endosperm is syncytial and proliferates rapidly through multiple rounds of mitosis in the absence of cytokinesis and cell wall formation. This stage of endosperm development is important in determining seed viability and size. To identify genes involved in syncytial endosperm development, we analyzed the endosperm transcriptome, obtained using laser capture microdissection of developing seeds at four days after pollination. Our results support the idea that similar sets of genes are required for conventional somatic mitosis with cytokinesis and syncytial proliferation. Furthermore, we identify cytoskeleton associated genes that may act to facilitate syncytial development thereby providing an important resource for further characterization of the processes involved in syncytial endosperm development.Seed development begins with double fertilization where the haploid egg cell and the double haploid central cell are both fertilized by haploid sperm cells contributed from a single pollen grain. This generates the diploid embryo and the triploid endosperm, respectively. The embryo and the endosperm grow rapidly in a coordinated manner that is influenced by the surrounding maternal integument tissues that later form the seed coat. During these early stages, maternal resources are used for the rapid cell division and growth that occurs as seed tissues form and develop. In many plant species, including Arabidopsis, the triploid primary endosperm nucleus undergoes several rounds of free-nuclear division, growing rapidly as a syncytium. The organization of microtubule arrays during this early stage of endosperm development is markedly different from those found in vegetative tissues.1 During the syncytial phase, interphase microtubules emanate from the nucleus into the cytoplasm and this nucleus-based radial microtubule system aids in positioning of nuclei and the designation of cytoplasm into nucleo-cytoplasmic domains. At about six days after pollination, cell wall deposition is initiated by the formation of phragmoplasts at the margins of nucleocytoplasmic domains. Interestingly, interzonal arrays which resemble phragmoplasts form between nuclei after karyokinesis, but they never participate in cell plate formation.2 This unusual form of cellular development is conducive to a rapid cellular proliferation; however, the molecular mechanisms that underlie the uncoupling of cell wall formation from mitosis remain elusive.To gain further insights into the early stages of endosperm biology, we have used laser capture microdissection to obtain RNA from syncytial stage endosperm at four days after pollination. At this stage of development, the endosperm has undergone six to seven rounds of nuclear division (~200 nucleocytoplasmic domains) but has not cellularized and the embryo is at the globular phase. Endosperm RNA was amplified using two-round in vitro transcription, then labeled and used, along with similarly treated silique amplified RNA, to probe long oligonucl...

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Evaluation of Global RNA Amplification and Its Use for High-Throughput Transcript Analysis of Laser-Microdissected Endosperm

Cited by 23 publications

References 74 publications

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

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

Identification of cytoskeleton-associated genes expressed during Arabidopsis syncytial endosperm development

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