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In praise of arrays.

Ying L, Sarwal M - Pediatr. Nephrol. (2008)

Bottom Line: Microarray technologies have both fascinated and frustrated the transplant community since their introduction roughly a decade ago.Fascination arose from the possibility offered by the technology to gain a profound insight into the cellular response to immunogenic injury and the potential that this genomic signature would be indicative of the biological mechanism by which that stress was induced.Frustrations have arisen primarily from technical factors such as data variance, the requirement for the application of advanced statistical and mathematical analyses, and difficulties associated with actually recognizing signature gene-expression patterns and discerning mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Stanford University, 300 Pasteur Drive, Stanford, CA 94305, USA.

ABSTRACT
Microarray technologies have both fascinated and frustrated the transplant community since their introduction roughly a decade ago. Fascination arose from the possibility offered by the technology to gain a profound insight into the cellular response to immunogenic injury and the potential that this genomic signature would be indicative of the biological mechanism by which that stress was induced. Frustrations have arisen primarily from technical factors such as data variance, the requirement for the application of advanced statistical and mathematical analyses, and difficulties associated with actually recognizing signature gene-expression patterns and discerning mechanisms. To aid the understanding of this powerful tool, its versatility, and how it is dramatically changing the molecular approach to biomedical and clinical research, this teaching review describes the technology and its applications, as well as the limitations and evolution of microarrays, in the field of organ transplantation. Finally, it calls upon the attention of the transplant community to integrate into multidisciplinary teams, to take advantage of this technology and its expanding applications in unraveling the complex injury circuits that currently limit transplant survival.

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Schematic representation of DNA microarray technology. Total RNA is first isolated from the samples of interest; this test RNA and a reference RNA are then differentially labeled with fluorescent dyes and then competitively hybridized onto a printed DNA microarray. Images that are generated are then scanned, and the resulting fluorescence intensities are used for further data analysis. IH immunohistochemistry, SNP single nucleotide polymorphism, SAM significance analysis of microarrays, PAM prediction analysis of microarrays, BRB Biometric Research Branch, GO Gene ontology, IPA Ingenuity Pathway Analysis, KEGG Kyoto Encyclopedia of Gene and Genomes
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Fig1: Schematic representation of DNA microarray technology. Total RNA is first isolated from the samples of interest; this test RNA and a reference RNA are then differentially labeled with fluorescent dyes and then competitively hybridized onto a printed DNA microarray. Images that are generated are then scanned, and the resulting fluorescence intensities are used for further data analysis. IH immunohistochemistry, SNP single nucleotide polymorphism, SAM significance analysis of microarrays, PAM prediction analysis of microarrays, BRB Biometric Research Branch, GO Gene ontology, IPA Ingenuity Pathway Analysis, KEGG Kyoto Encyclopedia of Gene and Genomes

Mentions: In organ transplantation, a prospective study of the gene-expression profile of graft injury usually involves sample collection from tissue biopsy, blood and biofluids, such as urine, bile, or broncho-alveolar lavage, taken before, during, and after injury. A schematic presentation of microarray study is shown in Fig. 1. All collected samples are then subject to standardized protocols for RNA extraction [1]. Routinely, one assesses RNA quality control by looking for ribosomal RNA ratios of 260/280 > 2. The integrity of the RNA can also be assessed with Agilent 2100 Bioanalyzer, using RNA Nano Chips (Agilent Technologies), where the degradation of RNA can be determined by RNA integrity number (RIN) [2, 3]. RNA amplification techniques are often required for microarray analysis and are related to downstream genetic analyses when small sample input is used as starting material. Linear RNA amplification is a strategy that has been used successfully to generate adequate input RNA for molecular profiling studies. One method of linear amplification, termed amplified antisense RNA (aRNA) amplification [4], utilizes a T7 RNA polymerase-based amplification procedure that allows quantitation of relative gene expression levels from small tissue samples. With modification of the classic Eberwine method, Wang et al. [5] exploited a template switching effect at the 5′ end of the mRNA transcript to ensure the synthesis of full-length double-stranded cDNA. The most common aspects arising from the use of sample amplification, irrespective of whether the method confers linear or exponential amplification, include amplification efficiency, 3′ bias and length of aRNA/cDNA products, reproducibility, fidelity of maintaining relative transcript abundance, benefits of the use of amplified material versus non-amplified material, and disadvantages with amplification procedures [6].Fig. 1


In praise of arrays.

Ying L, Sarwal M - Pediatr. Nephrol. (2008)

Schematic representation of DNA microarray technology. Total RNA is first isolated from the samples of interest; this test RNA and a reference RNA are then differentially labeled with fluorescent dyes and then competitively hybridized onto a printed DNA microarray. Images that are generated are then scanned, and the resulting fluorescence intensities are used for further data analysis. IH immunohistochemistry, SNP single nucleotide polymorphism, SAM significance analysis of microarrays, PAM prediction analysis of microarrays, BRB Biometric Research Branch, GO Gene ontology, IPA Ingenuity Pathway Analysis, KEGG Kyoto Encyclopedia of Gene and Genomes
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2719727&req=5

Fig1: Schematic representation of DNA microarray technology. Total RNA is first isolated from the samples of interest; this test RNA and a reference RNA are then differentially labeled with fluorescent dyes and then competitively hybridized onto a printed DNA microarray. Images that are generated are then scanned, and the resulting fluorescence intensities are used for further data analysis. IH immunohistochemistry, SNP single nucleotide polymorphism, SAM significance analysis of microarrays, PAM prediction analysis of microarrays, BRB Biometric Research Branch, GO Gene ontology, IPA Ingenuity Pathway Analysis, KEGG Kyoto Encyclopedia of Gene and Genomes
Mentions: In organ transplantation, a prospective study of the gene-expression profile of graft injury usually involves sample collection from tissue biopsy, blood and biofluids, such as urine, bile, or broncho-alveolar lavage, taken before, during, and after injury. A schematic presentation of microarray study is shown in Fig. 1. All collected samples are then subject to standardized protocols for RNA extraction [1]. Routinely, one assesses RNA quality control by looking for ribosomal RNA ratios of 260/280 > 2. The integrity of the RNA can also be assessed with Agilent 2100 Bioanalyzer, using RNA Nano Chips (Agilent Technologies), where the degradation of RNA can be determined by RNA integrity number (RIN) [2, 3]. RNA amplification techniques are often required for microarray analysis and are related to downstream genetic analyses when small sample input is used as starting material. Linear RNA amplification is a strategy that has been used successfully to generate adequate input RNA for molecular profiling studies. One method of linear amplification, termed amplified antisense RNA (aRNA) amplification [4], utilizes a T7 RNA polymerase-based amplification procedure that allows quantitation of relative gene expression levels from small tissue samples. With modification of the classic Eberwine method, Wang et al. [5] exploited a template switching effect at the 5′ end of the mRNA transcript to ensure the synthesis of full-length double-stranded cDNA. The most common aspects arising from the use of sample amplification, irrespective of whether the method confers linear or exponential amplification, include amplification efficiency, 3′ bias and length of aRNA/cDNA products, reproducibility, fidelity of maintaining relative transcript abundance, benefits of the use of amplified material versus non-amplified material, and disadvantages with amplification procedures [6].Fig. 1

Bottom Line: Microarray technologies have both fascinated and frustrated the transplant community since their introduction roughly a decade ago.Fascination arose from the possibility offered by the technology to gain a profound insight into the cellular response to immunogenic injury and the potential that this genomic signature would be indicative of the biological mechanism by which that stress was induced.Frustrations have arisen primarily from technical factors such as data variance, the requirement for the application of advanced statistical and mathematical analyses, and difficulties associated with actually recognizing signature gene-expression patterns and discerning mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Stanford University, 300 Pasteur Drive, Stanford, CA 94305, USA.

ABSTRACT
Microarray technologies have both fascinated and frustrated the transplant community since their introduction roughly a decade ago. Fascination arose from the possibility offered by the technology to gain a profound insight into the cellular response to immunogenic injury and the potential that this genomic signature would be indicative of the biological mechanism by which that stress was induced. Frustrations have arisen primarily from technical factors such as data variance, the requirement for the application of advanced statistical and mathematical analyses, and difficulties associated with actually recognizing signature gene-expression patterns and discerning mechanisms. To aid the understanding of this powerful tool, its versatility, and how it is dramatically changing the molecular approach to biomedical and clinical research, this teaching review describes the technology and its applications, as well as the limitations and evolution of microarrays, in the field of organ transplantation. Finally, it calls upon the attention of the transplant community to integrate into multidisciplinary teams, to take advantage of this technology and its expanding applications in unraveling the complex injury circuits that currently limit transplant survival.

Show MeSH
Related in: MedlinePlus