Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.
Anal Biochem
; 427(2): 178-86, 2012 Aug 15.
Article
en En
| MEDLINE
| ID: mdl-22617801
Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ARN Mensajero
/
Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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Transcripción Reversa
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Análisis de la Célula Individual
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
/
Male
Idioma:
En
Revista:
Anal Biochem
Año:
2012
Tipo del documento:
Article
Pais de publicación:
Estados Unidos