Selection of reference genes for normalization of quantitative real-time PCR in cell cultures of Cyclamen persicum

ABSTRACT

As a prerequisite for gene expression analyses in cell cultures of the ornamental crop Cyclamen persicum basic parameters for quantitative real-time polymerase chain reaction (qRT-PCR) have been established including the selection of reference genes using the software tools ‘geNorm’ and ‘NormFinder’. Five potential reference genes have been tested (elongation factor tu (Ef-Tu), putative ABC transporter ATPase, putative conserved oligomeric Golgi (COG) complex component, V-ATPase G subunit 1 and Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4)). ‘NormFinder’ as well as ‘geNorm’ identified Ef-Tu to be the least stable reference gene while the ranking of the most stable genes differed depending on the algorithm. According to ‘NormFinder’ COG complex component displayed the most stable expression whereas ‘geNorm’ indicated V-ATPase G subunit 1 and a putative ABC transporter ATPase to be the most reliable reference genes. Hence, we concluded to use a normalization factor calculated from the four reference genes V-ATPase G subunit 1, ABC transporter ATPase, Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4) and COG complex component for normalization of qRT-PCR in cell cultures of Cyclamen persicum.