Abstract
The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene-specific, unbiased transcriptome wide amplification accurately maintains proportionality among all RNA species within a given specimen. This allows the utilization of clinical material obtained with minimally invasive methods such as fine needle aspirates (FNA) or cytological washings for high throughput functional genomics studies. This review provides a comprehensive and updated discussion of the literature in the subject and critically discusses the main approaches, the pitfalls and provides practical suggestions for successful unbiased amplification of the whole transcriptome in clinical samples.
Highlights
Quantification of gene expression is a powerful tool for the global understanding of the biology underlying complex pathophysiological conditions
While the expression of a single or a limited number of genes can be readily estimated using minimum amount of total or messenger RNA from experimental or clinic samples, gene profiling requires large amount of RNA which can only be generated from global RNA amplification when using often limited amount clinical material
At least 50 – 100 μg of total RNA (T-RNA) or 2 – 5 μg poly(A)+ RNA are generally necessary for global transcript analysis studies though efforts to enhance signal intensity and fluorochrome incorporation have reduced the amount of total RNA needed for array analysis to 1–5 ug [13]
Summary
Quantification of gene expression is a powerful tool for the global understanding of the biology underlying complex pathophysiological conditions. In Van Gelder and Eberwine's T7 based RNA amplification [28], the amount of oligo dT-T7 primer used in the first strand cDNA synthesis can affect the amplified RNA in quantity and quality. The singularity of this approach resides in the utilization of a DNA polymerase blocker at the 3' of the oligo dA-T7 primer which prevents the elongation of second strand cDNA synthesis while priming for the elongation of the double stranded promoter In this fashion, only sense amplification can be achieved by the presence of the 5' ds-T7 promoter followed by single strand cDNA templates. The utilization of DNA polymerase with proofreading function could eradicate errors created in the cDNA amplification [64] This approach preserves the relative abundance of transcript [65] and it may outperform IVT when less than 50 ng of input RNA are available as starting material [66,67]. Centrifuge slide at 80–100 g for 3 min. (Slide can be put in slide rack on microplate carriers or in 50 ml conical tube and centrifuged in swinging-bucket rotor.)
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