Analysis of fungal gene expression by Real Time quantitative PCR.
Methods Mol Biol
; 638: 103-14, 2010.
Article
em En
| MEDLINE
| ID: mdl-20238263
ABSTRACT
The Real-Time quantitative PCR (qPCR) method has become central for the quantification of gene expression as well as other applications. The major advantages of qPCR are the utilization of small amount of template, high sensitivity and the ability to detect products during the reaction. After selecting qPCR among other options (northern blot, semi-quantitative PCR), one should consider several factors. The first and critical step in qPCR of fungi is the selection of an appropriate growth medium and RNA extraction method, which will avoid accumulation of inhibitors. In this chapter, we focus on detection of the accumulating product with the Syber Green dye, but other detection technologies, such as hybridization probes, might be considered as well. Accurate qPCR analysis with Syber Green depends mainly on optimal PCR reaction, and therefore it is important to design primers that will avoid formation of interfering structures. It is possible to use absolute quantification of the template in the sample, or to conduct a relative analysis, as described in this protocol. In the relative analysis method, expression of the gene of interest is compared with expression of a reference gene. According to our experience as well as according to the literature, it is recommended to use at least three reference genes in order to obtain reliable results.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
RNA Fúngico
/
Expressão Gênica
/
Reação em Cadeia da Polimerase
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Botrytis
/
Genes Fúngicos
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2010
Tipo de documento:
Article
País de afiliação:
Israel