RNA amplification strategies for small sample populations

Abstract

Advances in high throughput cloning strategies have led to sequencing of the human genome as well as progress in the sequencing of the genome of several other species. Consequently, the field of molecular genetics is blossoming into a multidisciplinary entity that is revolutionizing the way researchers evaluate a myriad of critical concepts such as development, homeostasis, and disease pathogenesis. There is tremendous interest in the quantitative assessment of tissue-specific expression of both newly identified and well characterized specific genes and proteins. At present, an ideal approach is to assess gene expression in single elements recorded physiologically in living preparations or by immunocytochemical or histochemical methods in fixed cells in vitro or in vivo. The quantity of RNA harvested from individual cells is not sufficient for standard RNA extraction methods. Therefore, exponential polymerase-chain reaction based analyses, and linear RNA amplification including amplified antisense RNA amplification and a newly developed terminal continuation RNA amplification methodology have been developed for use in combination with microdissection procedures and cDNA/oligonucleotide microarray platforms. RNA amplification is a series of intricate procedures to amplify genetic signals from minute quantities of starting materials for microarray analysis and other downstream genetic methodologies. RNA amplification procedures effectively generate quantities of RNA through in vitro transcription. The present report illustrates practical usage of RNA amplification technologies within the context of regional, population cell, and single cell analyses in the brain.