Global amplification of mRNA by template-switching PCR: linearity and application to microarray analysis

Abstract

Conventional approaches to target labelling for expression microarray analysis typically require relatively large amounts of total RNA, a serious limitation when the sample available is small. Here we explore the cycle-dependent ampli®cation characteristics of Template-Switching PCR and validate its use for microarray target labelling. TS-PCR iden-ti®es up to 80% of the differentially expressed genes identi®ed by direct labelling using 30-fold less input RNA for the ampli®cation, with the equivalent of 1000-fold less starting material being used for each hybridisation. Moreover, the sensitivity of microarray experiments is increased considerably, allowing the identi®cation of differentially expressed transcripts below the level of detection using targets prepared by direct labelling. We have also validated the ®delity of ampli®cation and show that the ampli®ed material faithfully represents the starting mRNA population. This method outperforms conventional labelling strategies, not only in terms of sensitivity and the identi®cation of differentially expressed genes, but it is also faster and less labour intensive than other ampli®cation protocols.

Inflammatory Gene Profiles in Gastric Mucosa during Helicobacter pylori Infection in Humans

“…Selected genes with a low level of expression, but with a proven biological relevance, should therefore be tested by real-time PCR. Alternatively, amplification of mRNA by template-switching PCR before array hybridization may allow identification of these genes (48).…”

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

“…Several methods and commercial kits for RNA amplification, that maintain the relative representation of RNA species, have been developed and can be used specifically for this purpose (Brandt 2005). The most common method is based on in vitro transcription (IVT) (Petalidis et al 2003). This linear amplification involves producing double-stranded cDNA with a T7 priming sequence at the 3?…”

Human Naive CD8 T Cells Down-Regulate Expression of the WNT Pathway Transcription Factors Lymphoid Enhancer Binding Factor 1 and Transcription Factor 7 (T Cell Factor-1) following Antigen Encounter In Vitro and In Vivo

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