Global amplification of mRNA by template-switching PCR: linearity and application to microarray analysis.
Nucleic Acids Res
; 31(22): e142, 2003 Nov 15.
Article
en En
| MEDLINE
| ID: mdl-14602935
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 amplification characteristics of Template-Switching PCR and validate its use for microarray target labelling. TS-PCR identifies up to 80% of the differentially expressed genes identified by direct labelling using 30-fold less input RNA for the amplification, 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 identification of differentially expressed transcripts below the level of detection using targets prepared by direct labelling. We have also validated the fidelity of amplification and show that the amplified material faithfully represents the starting mRNA population. This method outperforms conventional labelling strategies, not only in terms of sensitivity and the identification of differentially expressed genes, but it is also faster and less labour intensive than other amplification protocols.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ARN Mensajero
/
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2003
Tipo del documento:
Article
País de afiliación:
Reino Unido