Expression profiling using human tissues in combination with RNA amplification and microarray analysis: Assessment of Langerhans cell histiocytosis

Texas Children's Cancer Center/Hematology Service, Baylor College of Medicine, Houston, Texas, USA.
Amino Acids (Impact Factor: 3.29). 06/2005; 28(3):279-90. DOI: 10.1007/s00726-005-0177-x
Source: PubMed


Advances in molecular genetics have led to sequencing of the human genome, and expression data is becoming available for many diverse tissues throughout the body, allowing for exciting hypothesis testing of critical concepts such as development, differentiation, homeostasis, and ultimately, disease pathogenesis. At present, an optimal methodology to assess gene expression is to evaluate single cells, either identified physiologically in living preparations, or by immunocytochemical or histochemical procedures in fixed cells in vitro or in vivo. Unfortunately, the quantity of RNA harvested from a single cell is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction (PCR) based analyses, and linear RNA amplification including amplified antisense (aRNA) RNA amplification and a newly developed terminal continuation (TC) RNA amplification methodology have been used in combination with microdissection procedures such as laser capture microdissection (LCM) to enable the use of microarray platforms within individual populations of cells obtained from a variety of human tissue sources such as biopsy-derived samples {including Langerhans cell histiocytosis (LCH)} as well as postmortem brain samples for high throughput expression profiling and related downstream genetic analyses.

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Available from: Stephen D Ginsberg, Apr 07, 2015
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    • "However, a two-step approach may be more expensive, and it is often unclear whether a reduction in Type I and Type II errors will occur due to gene coexpression and sample variation (Prolla, 2002, 2005). Another statistical approach is using finite mixture modeling with jackknife methods, bootstrap analysis, or related routines (Aittokallio et al., 2003; Blalock et al., 2003; McClain et al., 2005). In summary, data analysis is a complex and important step in microarray research due to a myriad of experimental and statistical factors arising from the analysis of a multitude of data points with relatively low sample numbers and few replicates. "
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    • "Moreover, amplified aRNA products tend not to be of full length (Ginsberg et al., 1999a; Kacharmina et al., 1999; Phillips and Eberwine, 1996). Although aRNA is a laborious and difficult procedure, we have generated successful results obtained from microaspirated cells from animal model and postmortem human brain tissues utilizing a wide variety of array platforms (Ginsberg et al., 1999a, 2000; Hemby et al., 2002, 2003; McClain et al., 2005). "
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    ABSTRACT: The combination of tissue microdissection protocols including discrete cell microaspiration and laser capture microdissection with high throughput gene expression profiling platforms such as cDNA microarrays and oligonucleotide microarrays enables the simultaneous assessment of many individual elements from a single cell or a population of homogeneous cells. This chapter outlines in detail the theoretical and practical background for selecting the appropriate tissues and conditions amenable to expression profiling. In addition, this report illustrates the usage of microdissection strategies and RNA amplification methodologies in concert with array technologies using tissues harvested from the central nervous system obtained from animal models of neurodegeneration and postmortem human brain tissues.
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